The 1999–2000 season was the 85th season of the Isthmian League, which is an English football competition featuring semi-professional and amateur clubs from London, East and South East England.
Aylesbury United, Boreham Wood and Walton & Hersham finished bottom of the table and relegated to Division One.
Chertsey Town and Leyton Pennant relegated to Division Two, while Leatherhead were reprieved after Hemel Hempstead were refused promotion from Division Two.
Wingate & Finchley finished in relegation zone along with Witham Town and Chalfont St Peter and returned to Division Three.
Southall resigned and joined the Combined Counties League.