The 1999–2000 season was the 97th for the Southern Football League.
Boston United won the Premier Division and earned promotion to the Football Conference.
Atherstone United, Gloucester City, Grantham Town and Rothwell Town were relegated from the Premier Division, whilst Folkestone Invicta, Moor Green, Fisher Athletic and Stafford Rangers were promoted from the Eastern and Western divisions, the former two as champions.
Fleet Town, Yate Town and Stourbridge were relegated to the eighth level whilst Raunds Town resigned from the league.
The Eastern Division consisted of 22 clubs, including 16 clubs from the previous season Southern Division and six new clubs: