Dark greenhouse culture is one of the main culture methods of the Chinese soft-shelled turtle, Pelodiscus sinensis. The present study found that the outputs of P. sinensis differed among five ponds with the same initial stocking density and management measures, with the output of pond B with maximum yield being 1.69 times higher than that of pond A with minimum yield. Therefore, the physicochemical factors and bacterial communities of the two ponds were monitored, analyzed and compared. The contents of total nitrogen, nitrite nitrogen, and dissolved oxygen in pond B were extremely significantly higher than those in pond A at the late stage of cultivation. Correspondingly, the microbial diversity and richness were also higher in pond B. Additionally, the higher relative abundances of Gemmatimonadetes, Firmicutes, Nitrosomonas, Saccharimonadales andAzospira in pond B conferred it higher nitrification and denitrification ability than pond A in the middle and late stage of cultivation. Pearson’s correlation coefficient showed that the Saccharimonadales andAzospira were correlated with total nitrogen, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. Our results provide a reference for the modulation of the water environment in the dark greenhouse mode that used for turtle cultivation.