Tire and road wear particles (TRWP) contain complex mixtures of chemicals and release them to the environment,and potential toxic effects of these chemicals still need to be characterized. We used a standardized surrogate for TRWP,cryogenically milled tire tread (CMTT), to isolate and evaluate effects of tire‐associated chemicals. We examined organicchemical mixtures extracted and leached from CMTT for the toxicity endpoints genotoxicity, estrogenicity, and inhibition ofbacterial luminescence. The bioassays were performed after chromatographic separation on high‐performance thin‐layerchromatography (HPTLC) plates. Extracts of CMTT were active in all three HPTLC bioassays with two estrogenic zones, twogenotoxic zones, and two zones inhibiting bacterial luminescence. Extracts of CMTT artificially aged with thermooxidationwere equally bioactive in each HPTLC bioassay. Two types of aqueous leachates of unaged CMTT, simulating eitherdigestion by fish or contact with sediment and water, contained estrogenic chemicals and inhibitors of bacterial lumines-cence with similar profiles to those of CMTT extracts. Of 11 tested tire‐associated chemicals, two were estrogenic, three weregenotoxic, and several inhibited bacterial luminescence. 1,3‐Diphenylguanidine, transformation products of N‐(1,3‐dimethylbutyl)‐N′‐phenyl‐p‐phenylenediamine, and benzothiazoles were especially implicated through comparison to HPTLCretention factors in the CMTT samples. Other bioactive bands in CMTT samples did not correspond to any target chemicals.Tire particles clearly contain and can leach complex mixtures of toxic chemicals to the environment. Although some knownchemicals contribute to estrogenic, genotoxic, and antibacterial hazards, unidentified toxic chemicals are still present anddeserve further investigation. Overall, our study expands the understanding of potential adverse effects from tire particlesand helps improve the link between those effects and the responsible chemicals.