The draw for the first round was made at the 2023 AGM held in Blackpool this weekend. The draw was carried out, Facilitated by League Vice-Chairman Geoff Wilkinson, witnessed by League Director Martin Fallon. The Member Clubs representatives drew the Balls out of the bag. The ties will be played on a date TBA. Sadly, holders Congleton Town won't be able to defend their trophy having been moved to the Midland League this season.
| Macron Challenge Cup First Round | ||
| Abbey Hulton United | v | Stafford Town |
| Alsager Town | v | Eccleshall |
| Atherton LR | v | Ashton Athletic |
| Burscough | v | AFC Blackpool |
| Charnock Richard | v | Euxton Villa |
| Cheadle Heath Nomads | v | Ellesmere Rangers |
| Cheadle Town | v | Sandbach United |
| Colne | v | Barnoldswick Town |
| Darwen | v | Padiham |
| Droylsden | v | Abbey Hey |
| Garstang | v | Squires Gate |
| Holker Old Boys | v | Daisy Hill |
| Irlam | v | South Liverpool |
| Litherland Remyca | v | Pilkington |
| Longridge Town | v | Ashton Town |
| Lower Breck | v | Glossop North End |
| Maine Road | v | Ashville |
| Market Drayton Town | v | Stockport Georgians |
| New Mills | v | Bacup Borough |
| Ramsbottom United | v | Nelson |
| Route One Rovers | v | Steeton |
| Runcorn Town | v | FC St Helens |
| Shelley | v | Ilkley Town |
| Skelmersdale United | v | Kendal Town |
| Stockport Town | v | Brocton |
| Winsford United | v | Chadderton |
| Wythenshawe | v | Cammell Laird 1907 |
| Byes: AFC Liverpool, Bury, Prestwich Heys | ||
| West Didsbury & Chorlton, Wythenshawe Town | ||
