Despite the higher potential for eutrophication of dissolved organic nitrogen (DON) than dissolved inorganic nitrogen, the influence of the types of external carbon sources on DON remains largely unknown. This study investigated the effect of external carbon source type (acetate, glucose, methanol, and ethanol) on effluent DON characteristics in postdenitrifying moving bed biofilm reactors (MBBRs) from chemical molecular and microbial insights. The results showed that the effluent DON concentration in postdenitrifying MBBRs fed different carbon source types followed the order of ethanol > glucose > methanol > acetate. Interestingly, the acetate-fed postdenitrifying MBBR effluent DON contained a less labile molecular composition (5.03%), indicating that these effluent DON are not prone to stimulate algae production in receiving waters. Principal component analysis demonstrated that the produced DON instead of removed and/or resisted DON during the denitrification process mainly contributed to the differences in DON molecular composition and bioavailability in effluent. The key microbes, including Proteobacteria, Chloroflexi, etc. and amino acid metabolism were potentially responsible for the produced DON. Overall, this study demonstrated that MBBRs used for postdenitrification could be optimized to minimize eutrophication effects by accounting for optimal carbon sources and that acetate is recommended as the preferred external carbon source in wastewater treatment using postdenitrifying MBBRs.