2016/09/03 | Chesham United | | 5-0 | | Saffron Walden Town | | | | |
| Harrow Borough | | 4-1 | | Sawbridgeworth Town | | | | |
| Heybridge Swifts | | 2-2 | | AFC Dunstable | | | | |
| Maldon & Tiptree | | 0-1 | | Biggleswade Town | | | | |
| Sun Postal FC | | 0-2 | | Hayes & Yeading | | | | |
| Hendon | | 5-2 | | Cheshunt | | | | |
| AFC Sudbury | | 6-0 | | Halstead Town | | | | |
| Kings Langley | | 6-1 | | Sporting Bengal | | | | |
| Cambridge City | | 3-1 | | Flackwell Heath | | | | |
| Harlow Town | | 3-1 | | Romford | | | | |
| Uxbridge | | 2-0 | | Needham Market | | | | |
| Chalfont St Peter | | 0-3 | | Potters Bar Town | | | | |
| Felixstowe & Walton | | 2-1 | | Tilbury FC | | | | |
| Hanwell Town | | 1-0 | | Enfield Town | | | | |
| Kempston Rovers | | 3-2 | | Harefield United | | | | |
| Coalville Town | | 2-1 | | Redditch United | | | | |
| Mickleover | | 3-2 | | Spalding United | | | | |
| Hinckley Leicester Road | | 0-0 | | Kirby Muxloe | | | | |
| Highgate United | | 3-1 | | Leamington | | | | |
| Lincoln United | | 6-1 | | Dunkirk | | | | |
| Halesowen Town | | 3-0 | | Coleshill Town | | | | |
| Deeping Rangers | | 2-6 | | Gresley Rovers | | | | |
| Romulus | | 1-1 | | Hereford | | | | |
| Hadley FC | | 1-0 | | London Colney | | | | |
| Haringey Borough | | 0-3 | | Witham Town | | | | |
| Brightlingsea Regent | | 1-2 | | Billericay Town | | | | |
| Stafford Rangers | | 1-2 | | Kidsgrove Athletic | | | | |
| Eynesbury Rovers | | 1-3 | | Sutton Coldfield Town | | | | |
| Stanway Rovers | | 0-1 | | Barton Rovers | | | | |
| Canvey Island | | 2-1 | | Dunstable Town | | | | |
| Eastbourne United | | 0-3 | | Highworth Town | | | | |
| Faversham Town | | 1-0 | | Cray Wanderers | | | | |
| Herne Bay | | 3-1 | | Ashford Town (MIDDX) | | | | |
| CB Hounslow United | | 1-3 | | Metropolitan Police | | | | |
| Brimscombe & Thrupp | | 3-0 | | Peacehaven & Telscombe | | | | |
| Staines Town | | 4-0 | | Godalming Town | | | | |
| Pagham FC | | 0-3 | | Dulwich Hamlet | | | | |
| Arundel | | 1-5 | | Egham Town | | | | |
| VCD Athletic | | 4-1 | | Kingstonian | | | | |
| Slough Town | | 6-1 | | Chipstead | | | | |
| Wantage Town | | 1-1 | | Beaconsfield | | | | |
| Lewes | | 0-0 | | Sevenoaks Town | | | | |
| Worthing | | 3-3 | | Carshalton Athletic | | | | |
| Andover | | 0-5 | | Farnborough | | | | |
| Fleet Town | | 1-1 | | Slimbridge | | | | |
| AFC Rushden & Diamonds | | 2-0 | | Hornchurch | | | | |
| Lowestoft Town | | 0-2 | | Histon | | | | |
| Walthamstow FC | | 2-1 | | Stotfold | | | | |
| Leiston | | 4-1 | | Grays Athletic | | | | |
| Hitchin Town | | 4-2 | | Biggleswade Utd | | | | |
| Waltham Abbey | | 0-5 | | Wingate & Finchley | | | | |
| Colliers Wood | | 2-0 | | Eastbourne Town | | | | |
| Walton Casuals | | 3-2 | | Greenwich Borough | | | | |
| Burgess Hill Town | | 2-1 | | Ashford United | | | | |
| Camberley Town | | 0-2 | | Hastings United | | | | |
| East Preston | | 1-4 | | Merstham | | | | |
| Hythe Town | | 1-0 | | Leatherhead | | | | |
| Westfield FC | | 4-0 | | St. Ives Town | | | | |
| St Neots Town | | 1-1 | | Stamford | | | | |
| Brislington FC | | 5-2 | | Hamworthy United | | | | |
| Rugby Town FC | | 1-0 | | Corby Town | | | | |
| Bideford | | 1-2 | | AFC Portchester | | | | |
| Dorchester | | 0-3 | | Banbury Utd | | | | |
| Chippenham Town | | 9-0 | | Moneyfields | | | | |
| Basingstoke Town | | 4-0 | | Bemerton Heath Harlequins | | | | |
| Dereham Town | | 2-1 | | Holbeach United | | | | |
| Coventry United | | 0-2 | | Bedworth United | | | | |
| Ashton United | | 0-3 | | Nantwich Town | | | | |
| Brighouse Town | | 0-3 | | Lancaster City | | | | |
| Washington FC | | 2-5 | | Shildon AFC | | | | |
| South Park FC | | 2-1 | | Dorking Wanderers | | | | |
| Ascot United FC | | 2-2 | | Tonbridge Angels | | | | |
| Cadbury Heath | | 3-0 | | Plymouth Parkway | | | | |
| Gillingham | | 0-2 | | Cirencester Town | | | | |
| Bognor Regis Town | | 5-1 | | Guildford City | | | | |
| Ramsgate | | 1-2 | | Whyteleafe | | | | |
| Folkestone | | 3-1 | | North Greenford | | | | |
| Corinthian-Casuals | | 2-3 | | North Leigh | | | | |
| Thamesmead Town | | 1-0 | | Chertsey Town | | | | |
| Taunton Town | | 2-0 | | Cinderford Town | | | | |
| Alresford Town | | 1-1 | | Fareham Town | | | | |
| Salisbury City | | 2-0 | | Frome Town FC | | | | |
| Portishead Town | | 1-4 | | Swindon Supermarine | | | | |
| Sholing | | 0-2 | | Havant & Waterlooville | | | | |
| Winchester City | | 4-2 | | Street FC | | | | |
| Barnstaple Town | | 0-4 | | Merthyr Town | | | | |
| Blyth Spartans | | 3-1 | | Frickley Athletic | | | | |
| Hyde United | | 1-0 | | Colne FC | | | | |
| Hednesford Town | | 1-2 | | Belper Town | | | | |
| King´s Lynn | | 6-1 | | Brigg Town | | | | |
| Leek Town | | 2-3 | | Kettering Town | | | | |
| Radcliffe FC | | 3-5 | | Spennymoor Town | | | | |
| Trafford | | 5-2 | | Cheadle Town | | | | |
| Workington | | 3-0 | | Warrington Town | | | | |
| AFC Mansfield | | 2-1 | | Stratford Town | | | | |
| Rushall Olympic | | 2-2 | | Soham Town | | | | |
| Peterborough Sports | | 1-3 | | Stourbridge FC | | | | |
| Ashby Ivanhoe | | 0-6 | | Ilkeston FC | | | | |
| Evesham United | | 2-2 | | Barwell FC | | | | |
| Chasetown | | 1-1 | | Grantham Town | | | | |
| Matlock Town | | 4-2 | | Heanor Town | | | | |
| Burscough | | 4-3 | | Maine Road | | | | |
| Pontefract Collieries | | 2-3 | | Tadcaster Albion | | | | |
| Marske United | | 0-2 | | Marine AFC | | | | |
| Newcastle Benfield | | 0-2 | | Bridlington Town | | | | |
| Sheffield FC | | 0-3 | | Farsley Celtic | | | | |
| Whitby Town | | 3-3 | | Winsford United | | | | |
| Bishop Auckland | | 5-1 | | Ossett Albion | | | | |
| Morpeth Town | | 4-1 | | Colwyn Bay | | | | |
| Harrogate Railway | | 0-5 | | Consett AFC | | | | |
| Witton Albion | | 3-0 | | Buxton | | | | |
| Billingham Town | | 2-2 | | Ossett Town | | | | |
| Squires Gate | | 2-5 | | Worksop Parramore | | | | |
| Ashton Athletic | | 7-2 | | Mossley | | | | |
| Dunston UTS | | 2-2 | | Skelmersdale United | | | | |
2016/09/06 | Weymouth | | 2-1 | | Paulton Rovers | | | | |