Microplastics are pervasive contaminants in aquatic environments and can act as vectors for environmental contaminants by adsorbing them onto their surfaces, thereby altering contaminant transport, bioavailability, and biological effects. Tetracycline (TC), one of the most frequently detected antibiotics in aquatic environments, readily adsorbs onto microplastic surfaces, potentially resulting in carrier-mediated exposure to microorganisms. Although previous studies have primarily examined the combined effects of microplastics and antibiotics under co-exposure conditions, the biological responses elicited by antibiotic-adsorbed microplastics remain largely unexplored. This study investigated the physiological and molecular responses of Escherichia coli DH5α following exposure to TC-adsorbed polystyrene (PS) microplastics using physiological, biochemical, and transcriptomic analyses. Exposure to TC-adsorbed PS caused greater inhibition of bacterial viability than exposure to PS or TC alone and was accompanied by increased oxidative stress and membrane damage, demonstrating that adsorption of TC onto PS altered bacterial physiological responses. Transcriptomic analysis was performed to identify differentially expressed genes and biological pathways associated with exposure to TC-adsorbed PS, providing mechanistic insight into the molecular basis of these responses. Collectively, these findings demonstrate that contaminant adsorption onto microplastics can induce carrier-mediated toxicity that cannot be fully explained by simple co-exposure, highlighting the importance of considering contaminant-loaded microplastics in ecological risk assessments.