The enhanced adsorption of pollutants on biofilm-developed microplastics has been proved in many studies, but the ecotoxicological effects of biofilm-developed microplastics on organisms are still unclear. In this study, adult zebrafish were exposed to original microplastics, biofilm-developed microplastics, original microplastics absorbed with oxytetracycline (OTC), and biofilm-developed microplastics absorbed with OTC for 30 days. The intestinal histological damage, intestinal biomarker response, gut microbiome and antibiotic resistance genes (ARGs) profile of zebrafish were measured to explore the roles of biofilm in the effects of microplastics. The results showed that biofilm-developed microplastics significantly increased the number of goblet cells in intestinal epithelium compared with the control group. The biofilm-developed microplastics also induced the oxidative response in the zebrafish intestines, and biofilm changed the response mode in the combined treatment with OTC. Additionally, the biofilm-developed microplastics caused intestinal microbiome dysbiosis, and induced the abundance of some pathogenic genera increasing by several times compared with the control group and the original microplastics treatments, regardless of OTC adsorption. Furthermore, the abundance of ARGs in biofilm-developed microplastics increased significantly compared with the control and the original microplastic treatments. This study emphasized the significant influence and unique role of biofilm in microplastic studies.