Microplastics (MPs) have introduced new surfaces for biofilm development and gene exchange among bacteria. We investigated Tire Wear Particles (TWPs) for their involvement in horizontal gene transfer (HGT), particularly in relation to associated metals in the matrices of TWPs. We employed red-fluorescently tagged E. coli strain as a donor with green-fluorescently tagged, broad-host-range plasmid pKJK5, resistant to trimethoprim. As a recipient, we utilized Pseudomonas sp. and a natural lake microbial community. HGT activity on TWPs was determined and compared with that on polystyrene (PS) (with and without metals), and chitosan, which was used as a natural surface. Exposure to TWPs significantly enhanced HGT frequency of antibiotic resistance gene (ARG) from donor to recipient compared to PS and chitosan, and metals of TWPs further promoted HGT. HGT frequency on TWPs with Pseudomonas sp. was found to be 10⁻³ at 30 °C. in the lake community, it was similarly high already at 25 °C suggesting a higher permissiveness of the natural microbial community towards ARG at lower temperatures. This study sheds light on the potential impact of TWPs in promoting HGT, forming the basis for health risk assessments of TWPs and more generally of MP pollution in various aquatic ecosystems.