Antimicrobial resistance (AMR) is a critical threat to global health, with aquatic ecosystems playing a key role in the spread of antibiotic resistance genes (ARGs), especially those impacted by wastewater discharge. In this study, we investigate how water flow hydrodynamics influence ARG distribution and microbial community dynamics in river biofilms exposed to wastewater effluent. Experiments were performed under laboratory conditions using natural river biofilms in river flume systems that simulate static (S; stagnant pools or lake/pond-like water) and dynamic (D; flowing or river-like water) aquatic environments. High-throughput qPCR was used to analyze the presence and abundance of 36 marker genes for AMR, mobile genetic elements, and specific bacterial faecal pollution indicators. 16S rRNA gene sequencing tracked bacterial community dynamics over time. We observed distinct microbial community compositions in the S and D systems, with the D system exhibiting higher diversity. Across genes including intI1, mphA, aph3, aac(6′), blaCTX-M, ARG trajectories diverged by flow: D maintained or accumulated relative ARG abundances over time, whereas S exhibited progressive ARG loss. In contrast, the S system showed lower diversity and progressive ARG loss over time, emphasizing the role of water flow in maintaining ARG abundance within biofilm communities. Overall, these findings inform One Health and wastewater management by demonstrating that hydrodynamic conditions shape ARG trajectories, with dynamic flow promoting ARG persistence and slower loss compared to static conditions.