Cold-pressed Zanthoxylum schinifolium oil wastewater (ZOW), rich in organic pollutants, poses serious environmental challenges. This study examined ZOW treatment using a biochemical process, analyzing changes in activated sludge (AS) characteristics and microbial diversity to clarify pollutant removal pathways. Optimal parameters were established for biological treatment: a temperature of 30 °C, mixed liquor suspended solids (MLSS) of 4000 mg/L, initial pH of 7.5, rotational speed of 200 rpm, and a hydraulic retention time (HRT) of 48 h. For flocculation, 400 mg/L of polymeric aluminum sulfate and 60 mg/L of amphoteric polyacrylamide at pH 8 were identified as ideal. The biochemical process significantly reduced chemical oxygen demand (94.03 ± 0.31%), total phosphorus (93.33 ± 0.25%), total nitrogen (97.03 ± 0.26%), and turbidity (98.97 ± 0.02%). AS characterization showed enhanced sedimentation, larger particle size, increased extracellular polymer production indicating greater adsorption capacity, and higher biomass. 16S rRNA sequencing revealed Proteobacteria and Bacteroidota as dominant taxa (>
50%), with aerobic denitrifiers such as Chryseobacterium and Pseudomonas showing strong adaptation to ZOW. Functional annotation confirmed the AS community's efficacy in pollutant removal.