Abstract: High concentration of secondary hydrogen sulfide (sH2S) in thermal recovery reservoirs of Liaohe Oilfield, NE China was concluded to originate from thermochemical sulfate reduction (TSR), and no biotic source of H2S under abundant biomass has been reported in these presumed steam sterilized reservoirs ever before. In this study, we propose a new mechanism, biomass thermal decomposition for sulfur compounds (BTDS), to interpret the increasing of sH2S. Sulfur of cells’ dry weight took 0.20%–1.92% of the active strains isolated from the in-situ thermal recovery reservoirs of Liaohe Oilfield. When microbial organic sulfur compounds (MOSC) in biomass were exposed to injected steam, it resulted in the BTDS process. The isolated Bacillus subtilis D3 (G+) and Pseudomonas aeruginosa XJ14 (G−) were chosen to simulate this process. About 36% of sulfur in MOSC emitted as H2S in steam chamber by BTDS. The δ34S of H2S from produced gas ranged from 8.7‰ to 17.0‰, close to the δ34S of H2S 11.2‰ from BTDS simulation experiment. It provides new insight into the contribution and sulfur cycle made by subterranean microorganisms on H2S formation.