Sulfur-rich natural gas reservoirs have been discovered in the southwestern margin of the Qaidam Basin, marking a breakthrough in the Cenozoic natural gas exploration. 16S rRNA extraction and analysis were performed on crude oil samples from H2S-rich reservoirs at the Yuejin, Shizigou and Huatugou profiles to understand the sulfurous gas origination, which was also intergrated with carbon and hydrogen isotopes of alkane and sulfur isotopes of H2S collected from the Yingxiongling Area. Results show that microorganisms in crude oil can be classified into Proteobacteria, Planctomycetes, Firmicutes, Bacteroidetes, Haloanaerobe, etc., which can survive in hypersaline reservoirs. Methanogens and nitrate-reducing bacteria are popular in three profiles, while sulfate-reducing bacteria are abundant in Yuejin and Huatugou profiles, which contributed to methane and H2S in natural gas. The gas components and sulfur isotopes at the Yingxiongling Area show that the natural gas was mixed by coal-type gas and oil-type gas, which was primarily derived from thermal degradation, and natural gas from the Yuejin and Huatugou profiles also origniated from biodegradation. It agrees well with the tested microbial sequencing, i.e., H2S-rich natural gas from the Cenozoic reservoirs in the southwest margin of the Qaidam Basin was primarily thermal genesis, with microbial genesis of secondary importance.