The ever-increasing microplastics (MPs) and antibiotic-resistance genes (ARGs) in aquatic ecosystems has become a serious global challenging issue. However, the impact of different pollution sources on microbiome and antibiotic resistome in surface water (SW) and plastisphere (PS) remains largely elusive. Here, shotgun metagenomics was used to analyze microbiome structure and antibiotic resistome in SW and PS under the influence of different pollution sources. Pseudomonas were the most abundant genus, followed by Flavobacterium, Acinetobacter, Acidovorax, and Limnohabitans. However, their relative abundance varied significantly both across the sampling sites and habitats i.e. SW and PS (p <
0.05). Additionally, various ARGs were detected in SW and PS, with PS (372) having significantly more potential ARGs than SW (293). The results further showed significant variations in the relative abundance of potential pathogenic bacteria across the sampling sites and habitats (p <
0.05). Further moreover, significant differences were observed in antibiotic resistome risk scores, ARGs and MGEs across different habitats. Over all, this study suggests that pollution source and water quality parameters had a significant impact on microbiome composition and antibiotic resistome in SW and PS.