Ecological impacts of tire wear particles (TWPs) on microbial communities and biogeochemical cycles in freshwater remain largely unknown. Here, we conducted a microcosm experiment to investigate interactions between the overlying water and sediment without and with TWPs addition in a rural vs. urban lake system. Our results revealed the degree of change in microbial community diversity in water is higher than that in sediment following TWPs addition. For bacterial communities, TWPs addition changed their composition in the water, but only little in the sediment. For fungal communities, TWPs addition changed their composition both in water and sediments. Furthermore, in water, TWPs addition increased network complexity between bacteria-bacteria, fungi-fungi and bacteria-fungi in the urban system but reduced it in the rural one. In contrast, TWPs presence did not significantly change network complexity among microbial communities in the sediment of both lakes. Isotope labeling analysis uncovered that based on a short-term (6 hours) incubation experiment, TWPs addition did not significantly change carbon nor nitrogen cycling in the water. Yet, certain changes could be observed, especially in the long-term experiment (1 month), indicating that TWPs pollution has the potential to impact elemental cycling and thus ecosystem functions by altering microbial communities. Our results provide new insights into TWPs-induced ecological effects on microorganisms and potential biogeochemical consequences in a rural vs. urban lakes.