Tire and road wear particles (TRWP) account for an important part of the polymer particles released into the environment. There are scientific knowledge gaps as to the potential bioaccessibility of chemicals associated with TRWP to aquatic organisms. This study investigated the solubilization and bioaccessibility of seven of the most widely used tire-associated organic chemicals and four of their degradation products from cryogenically milled tire tread (CMTT) into fish digestive fluids using an in vitro digestion model based on Oncorhynchus mykiss. Our results showed that 0.06–44.1% of the selected compounds were rapidly solubilized into simulated gastric and intestinal fluids within a typical gut transit time for fish (3 h in gastric and 24 h in intestinal fluids). The environmentally realistic scenario of coingestion of CMTT and fish prey was explored using ground Gammarus pulex. Coingestion caused compound-specific changes in solubilization, either increasing or decreasing the compounds’ bioaccessibility in simulated gut fluids compared to CMTT alone. Our results emphasize that tire-associated compounds become accessible in a digestive milieu and should be studied further with respect to their bioaccumulation and toxicological effects upon passage of intestinal epithelial cells.