Perchlorate (ClO4-) mainly exists in the form of ammonium perchlorate in industrial production. However, the degradation mechanisms of different concentrations of ammonium nitrogen (NH4+-N) and ClO4- mixed pollutants in the environment are not well understood. This study aims to explore the potential of different types of carbon sources for ClO4- and NH4+-N biodegradation. Experimental results showed that the concentration and type of carbon sources are decisive to simultaneous removal of NH4+-N and ClO4-. Under condition of C(COD)/C(ClO4-) ratio of 21.15 ± 4.40, the simultaneously removal efficiency of ClO4- and NH4+-N in acetate (Ace) was relatively higher than that in methanol (Met). C(NH4+-N)/C(ClO4-) ratio of 9.66 ± 0.51 and C(COD)/C(ClO4-) ratio of 2.51 ± 0.87 promoted ClO4- reduction in glucose-C (Glu-C). However, high concentration of Glu could cause pH decrease (from 7.57 to 4.59), thereby inhibiting ClO4- reduction. High-throughput sequencing results indicated that Proteobacteria and Bacteroidetes have made a major contribution to the simultaneous removal of NH4+-N and ClO4-. They are two representative bacterial phyla for participating in both ClO4- reduction and denitrification. Notably, the abundance of main ClO4- degrading bacteria (such as Proteobacteria, Chloroflexi, and Firmicutes) significantly increased by 528.57 % in Glu-C. It can be inferred that the concentration of carbon source and NH4+-N were the most important factors determining the removal efficiency of ClO4- by influencing changes in the core microbial community. This study will provide new techniques and mechanistic insights for the simultaneous removal of mixed ClO4- and nitrogen pollutants, which can also provide theoretical support for innovation in future biological treatment processes.