Among non-exhaust emissions, road dust resuspension represents a rather important contribution to particulate matter in urban areas. This study aimed to achieve a chemical characterisation of road dust particulate matter (PM10) on two motorway sections, one rural and one urban, and to explore the related health and ecotoxicological risks. Measured PM10 dust loadings reached very low levels (0.66–1.49 mg m-2) compared to equivalent studies in other road environments in Portugal and other countries. Emission factors ranged from 33 to 62 mg veh−1 km−1. The carbonaceous content represented 14% of the total PM10 mass, whereas the highest contribution to the mass was given by mineral matter. Elements such as Si, Al, Ca, Fe and K accounted for almost three quarters of the total element mass for all samples, whilst Cu and Zn, mostly associated with brake and tyre wear, were the most enriched elements in relation to the soil composition. Nonetheless, Ti and Zr presented the highest non-carcinogenic risks for human health. Despite the low amounts of particulate matter in the aqueous solution, the ecotoxicological screening with the Aliivibrio fisheri bioluminescence inhibition bioassay allowed to classify the samples as toxic.