Substitute Armand Gnanduillet ended Southend's 100% record with an injury-time winner at the ProAct Stadium.
Gnanduillet headed home in a hectic finish which saw Luke Prosser twice denied from a corner at the other end.
Chesterfield had the better chances in the first half and eventually went in front when Conor Clifford fouled Gary Roberts, who curled home the free-kick.
Freddy Eastwood levelled from Kevan Hurst's pass, but it was not enough as Gnanduillet eluded his marker to score.
The victory lifted them up to second in the table behind Oxford United, while Southend slipped two places to fourth.
VIEW FROM THE DRESSING ROOM
Chesterfield manager Paul Cook told BBC Radio Sheffield:
"We didn't play well today, we must give great credit to Southend because I thought they were excellent.
"Certainly in the latter stages of the game I thought they were the team that looked more likely to win the game.
"Football's a funny game, you must give credit to the opposition. They scored a good equaliser, it was a fantastic finish and at that point I would have been happy to see the game end 1-1.
"We looked lethargic today and I don't know where it came from."
Chesterfield 01 Lee 03 Smith Booked 04 Hird 06 Cooper 25 Talbot 05 Morsy 10 O'Shea (Gnanduillet - 77' ) 11 Roberts 12 Ryan 15 Humphreys (Darikwa - 57' Booked ) 09 Richards (Doyle - 64' ) Substitutes 20 Chapman 08 Togwell 27 Edwards 07 Darikwa 16 Devitt 17 Doyle 21 Gnanduillet Southend 17 Bentley 02 White Booked 03 Coker 06 Prosser Booked 23 Thompson 11 Straker 12 Atkinson 16 Clifford Booked (Hurst - 60' ) 18 Leonard 07 Eastwood (Timlin - 83' ) 10 Corr (Cowan - 78' ) Substitutes 01 Smith 27 Banton 08 Timlin 14 Hurst 21 Payne 09 Reid 15 Cowan
Ref: Seb Stockbridge