Dissolved organic nitrogen (DON) represents a unique challenge in prechlorinated raw water distribution systems (PRWDSs) because of its contribution to the formation of harmful nitrogen-disinfection byproducts, influence upon biogeochemical processes, and unclear molecular characteristics. Here, Fourier transform ion cyclotron resonance mass spectrometry in combination with high-throughput sequencing was applied to elucidate the molecular changes of DON and biofilm microbial communities in a PRWDS in Yixing, China. Our study revealed that dynamic characteristics of DON are significantly correlated with the biofilm. The accumulation of refractory lignin-like compounds and CnHmOpN1 contributes to the higher recalcitrance molecular characteristics of DON in the effluent associated with Alphaproteobacteria, Planctomycetes, and Bacteroidetes. Additionally, with the help of prechlorination, the biofilm may change the DON characteristics and lead to higher oxygenation, higher m/z, and lower saturation during transportation. Despite the promotion of CnHmOpN1 and CnHmOpN3 at the early stage, we suggest that appropriate concentration of chlorine can add to the front end of raw water distribution pipes. Prechlorination may control the nitrification process and stabilize the rapid growth of diversity and concentration of low molecular weight DON, especially the refractory CnHmOpN1 in the effluent, which may help to improve treatment efficiency of drinking water treatment plants.