Buriram, who had not suffered defeat in the league since April, were four points clear heading into the midweek fixtures after Chonburi were held to a 1-1 draw at Suphanburi at the weekend.
However, the race for the title is now well and truly back in the balance - Sutthinan Pukhom scoring in the 89th minute to seal a 1-0 win over the defending champions.
Buriram will have the chance to bounce back when they face struggling Air Force Central on Saturday, before Chonburi continue their quest for top spot against Singhtarua a day later.
Muang Thong United also remain firmly in the hunt for the title after cruising to a 3-0 win over basement club Samut Songkhram.
Rock-bottom Samut had registered their first win in 11 top-flight attempts at the weekend but they were brushed aside with ease by Muang Thong, who are now just one point behind second-placed Chonburi.
Police United headed into the midweek round having drawn each of their last five matches.
However, a sequence of 10 matches without a win was brought to an end at Singhtarua thanks to a 62nd-minute strike from Michael Murcy.
The win lifts Police out of the relegation zone, while Air Force Central also boosted their chances of survival by beating Suphanburi 2-0.
Songkhla United remain mired in relegation trouble following a 1-0 defeat to TOT, while PTT Rayong held Sisaket to a goalless draw.
Bangkok United and BEC Tero Sasana also played out a 0-0 stalemate.
Elsewhere, Chiangrai United's winless league run stretched to five matches as they were defeated 3-1 by Army United, Chainat Hornbill and Ratchaburi drew 2-2, and a goal apiece in the opening six minutes saw Osotspa and Bangkok Glass share the spoils 1-1.
