This study aims to investigate the effects of long-term nicosulfuron residue on an herbicide factory ecosystem. High-throughput sequencing was used to investigate the environmental microbial community structure and interactions. The results showed that the main contributor to the differences in the microbial community structure was the sample type, followed by oxygen content, pH and nicosulfuron residue concentration. Regardless of the presence or absence of nicosulfuron, soil, sludge, and sewage were dominated by groups of Bacteroidetes, Actinobacteria, and Proteobacteria. Long-term exposure to nicosulfuron increased alpha diversity of bacteria and archaea but significantly decreased the abundance of Bacteroidetes and Acidobateria compared to soils without nicosulfuron residue. A total of 81 possible nicosulfuron-degrading bacterial genera, e.g., Rhodococcus, Chryseobacterium, Thermomonas, Stenotrophomonas, and Bacillus, were isolated from the nicosulfuron factory environmental samples through culturomics. The co-occurrence network analysis indicated that the keystone taxa were Rhodococcus, Stenotrophomonas, Nitrospira, Terrimonas, and Nitrosomonadaceae_MND1. The strong ecological relationship between microorganisms with the same network module was related to anaerobic respiration, the carbon and nitrogen cycle, and the degradation of environmental contaminants. Synthetic community (SynCom), which provides an effective top-down approach for the critical degradation strains obtained, enhanced the degradation efficiency of nicosulfuron. The results indicated that Rhodococcus sp. was the key genus in the environment of long-term nicosulfuron exposure. Display omitted •Nicosulfuron-degrading strains in chronically contaminated soil become dominant.•Long-term nicosulfuron residue increased the alpha diversity of soil microorganisms.•A synthetic community with Rhodococcus sp. had enhanced degradation efficiency.