Road dust is an important urban reservoir of potentially toxic elements (PTEs) and a relevant source of human exposure through resuspension and inhalation, particularly in large megacities. This study provides an integrated assessment of the seasonal variability, contamination levels, source identification, particle characteristics, lung bioaccessibility, and health risk of road dust in Mexico City, one of the world’s largest urban centers. A total of 74 road dust samples were collected during the dry and wet seasons, and V, Cr, Mn, Co, Ni, Cu, As, Cd, Sb, and Pb were analyzed by ICP–MS in the <20 µm fraction. Geochemical indices, spatial analysis, Pearson correlation, principal component analysis, SEM–EDS particle characterization, in vitro lung bioaccessibility (ALF), and human health risk models were applied. Sb, Cu, and Pb were identified as the most enriched elements, exceeded local background concentrations at all sampling sites. Spatial patterns revealed recurrent hotspots in the northern, northeastern, and central sectors of the city. SEM–EDS analyses showed that most particles belonged to the 2.5–5 µm equivalent-size class and included Fe-rich spherules, Pb-rich aggregates, silicate grains, and C-rich particles. Health risk assessment indicated acceptable risks for adults, whereas children exceeded the non-carcinogenic threshold (HI = 3.85–4.60) and slightly surpassed the upper acceptable carcinogenic risk level. Lung bioaccessibility results revealed low Pb solubility but high mobility of Ni and Cu, with some samples reaching complete dissolution under ALF conditions. These findings demonstrate that traffic-derived road dust represents a persistent urban exposure pathway in Mexico City and highlight the importance of integrating total concentrations, particle characteristics, and bioaccessibility data to improve environmental and health-risk assessments in urban environments.