Vehicular traffic generates the so-called Tire Road Wear Particles (TRWPs), debris produced by the wear of both tires and road pavements. Asphalts, in turn, can be modified with recycled rubber from End-of-Life Tires (ELTs) and polymers to improve their mechanical performance and reduce traffic noise. Therefore, the aim of this study was to determine whether the incorporation of recycled rubbers and polymers into asphalt mixtures affects the toxicity of obtained particles. Since TRWP toxicity is strongly influenced by environmental factors, such as road runoff, as well as by the wide plethora of chemicals derived from vehicular traffic, the particles used in this study were produced under controlled laboratory conditions to specifically investigate the effects of rubbers and plastics used in the asphalt mixtures. As these particles do not contain the fraction originating from tire tread abrasion, they are referred to as Road Wear Particles (RWPs). The resulting powders were characterized by Scanning Electron Microscopy (SEM) coupled with Energy-Dispersive X-ray Spectroscopy (EDS). Aqueous suspensions of RWPs were then prepared by stirring for 72 h, followed by a 48 h settling and subsequently characterized by Dynamic Light Scattering (DLS). Zebrafish larvae were exposed to 10 and 100 µg/L of RWP suspensions from 0 to 120 hours post-fertilization (hpf). Adverse effects were assessed using a comprehensive battery of biomarkers related to cellular stress, neuro-genotoxicity, heart rate alterations, and swimming behavior. All endpoints were integrated into the Biomarker Response Index (BRI) to obtain a toxicity scale of the tested materials. The main findings indicate that particles generated from modified asphalts do not exhibit a clear increase in toxicity, as particles derived from conventional asphalt frequently showed comparable toxic effects. These results are further supported by a previous study in which environmentally TRWPs were found to be more toxic when associated with conventional asphalt.