Rubberized materials, derived from End-of-Life Tires (ELTs), and synthetic polymers can be incorporated into asphalt mixtures to enhance durability and reduce traffic noise. However, the abrasion of tires on asphalt surfaces during traffic generates so-called Tire Road Wear Particles (TRWPs), which represent contaminants in both terrestrial and aquatic environments. The aim of this study was to compare the toxicity of TRWPs collected from rubberized, polymer-modified, and conventional asphalts. These contaminants were collected in December 2024 using a vacuum system along the 73 Senese Aretina State Road (Central Italy), which features different contemporary types of rubberized, polymer-modified, and conventional asphalts. The collected particles were characterized in terms of ultrastructure by Scanning Electron Microscopy (SEM/EDS), presence of microplastics by µFourier Transform Infrared Spectroscopy (µFT-IR), and occurrence of the additive Hexamethoxymethylmelamine (HMMM) to confirm the presence of tire-derived particles. Subsequently, TRWP suspensions (10 and 100 µg/L) were prepared by 72 h of stirring followed by 48 h of settling. The suspensions were characterized by Dynamic Light Scattering and Single Particle Extinction and Scattering (SPES) to obtain both the size and concentration of particles. In addition, a non-target chemical analysis was performed to characterize the main chemicals released by TRWPs in water. Chronic toxicity was evaluated in Danio rerio (zebrafish) larvae exposed from 0 to 120 hours post-fertilization (hpf). A wide battery of biomarkers of cellular stress, cellular damage, neuro-genotoxicity, as well as apical endpoints, such as heart rate and swimming behavior, was applied together with omics techniques, such as gel-free proteomics, metabolomics, and transcriptomics. This comprehensive approach, coupled with the integration of results in the Biomarker Response Index (BRI), suggests that the use of rubberized and polymer-modified materials in asphalt mixtures does not affect TRWP toxicity, highlighting, albeit slightly, a greater toxicity of TRWPs derived from conventional asphalt.