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. The league consisted of four divisions.
| Competition: | Isthmian League Premier Division |
| Season: | 1999–2000 |
| Winners: | Dagenham & Redbridge |
| Promoted: | Dagenham & Redbridge |
| Relegated: | Aylesbury United Boreham Wood Walton & Hersham |
| Matches: | 462 |
| Total Goals: | 1357 |
| Highest Attendance: | 5,518 – Aldershot Town – Farnborough Town, (27 December) |
| Attendance: | 248,459 |
| Average Attendance: | 538 (-5.8% to previous season) |
| Prevseason: | 1998–99 |
| Nextseason: | 2000–01 |
The Premier Division consisted of 22 clubs, including 19 clubs from the previous season and three new clubs:
Dagenham & Redbridge won the division and were promoted to the Football Conference. Aylesbury United, Boreham Wood and Walton & Hersham finished bottom of the table and relegated to Division One.
Before the start of the season Hampton changed name into Hampton & Richmond Borough.
| Competition: | Isthmian League Division One |
| Season: | 1999–2000 |
| Winners: | Croydon |
| Promoted: | Croydon Grays Athletic Maidenhead United |
| Relegated: | Chertsey Town Leyton Pennant |
| Matches: | 462 |
| Total Goals: | 1320 |
| Highest Attendance: | 919 – Bedford Town – Barton Rovers, (27 December) |
| Attendance: | 103,536 |
| Average Attendance: | 224 (+10.9% to previous season) |
| Prevseason: | 1998–99 |
| Nextseason: | 2000–01 |
Division One consisted of 22 clubs, including 17 clubs from the previous season and five new clubs:
Two clubs relegated from the Premier Division:
Three clubs promoted from Division Two:
Croydon won the division and returned to the Premier Division. Grays Athletic and Maidenhead United also get a promotion. Chertsey Town and Leyton Pennant relegated to Division Two, while Leatherhead were reprieved after Hemel Hempstead were refused promotion from Division Two.
| Competition: | Isthmian League Division Two |
| Season: | 1999–2000 |
| Winners: | Hemel Hempstead Town |
| Promoted: | Ford United Northwood |
| Relegated: | Chalfont St Peter Wingate & Finchley Witham Town |
| Matches: | 462 |
| Total Goals: | 1489 |
| Highest Attendance: | 464 – Northwood – Hemel Hempstead Town, (29 April) |
| Attendance: | 49,201 |
| Average Attendance: | 106 (-19.7% to previous season) |
| Prevseason: | 1998–99 |
| Nextseason: | 2000–01 |
Division Two consisted of 22 clubs, including 16 clubs from the previous season and six new clubs:
Three clubs relegated from Division One:
Three clubs promoted from Division Three:
Hemel Hempstead Town won the division, but were refused promotion due to ground grading. Northwood were promoted to Division One along with Ford United who get a second consecutive promotion. Wingate & Finchley finished in relegation zone along with Witham Town and Chalfont St Peter and returned to Division Three.
| Competition: | Isthmian League Division Three |
| Season: | 1999–2000 |
| Winners: | East Thurrock United |
| Promoted: | East Thurrock United Great Wakering Rovers Tilbury |
| Matches: | 420 |
| Total Goals: | 1326 |
| Highest Attendance: | 521 – Hertford Town – Ware, (27 December) |
| Attendance: | 34,483 |
| Average Attendance: | 82 (+7.9% to previous season) |
| Prevseason: | 1998–99 |
| Nextseason: | 2000–01 |
Division Three consisted of 21 clubs, including 17 clubs from the previous season and four new clubs:
Great Wakering Rovers debuted in the league and achieved promotion at the first attempt along with East Thurrock United and Tilbury. Southall resigned and joined the Combined Counties League.