Road traffic represents a major source of particulate matter (PM), which is considered the most harmful pollutant for humans. This work aimed to evaluate the toxic effects of polycyclic aromatic hydrocarbons (PAHs) and plasticisers extracted from PM emitted by traffic, including exhaust samples, samples collected in a road tunnel and simultaneously in a nearby urban background atmosphere, and samples from the wear of distinct types of brakes. Different biological endpoints were investigated after exposing human alveolar epithelium A549 cells to the organic extracts: viability, cell cycle alterations and levels of intracellular reactive oxygen species (ROS). The particulate extracts from the wear of low steel brake pads induced greater decreases in cell viability. It was observed that cytotoxicity is more dependent on the individual composition of PAHs than on the global concentration. Exhaust samples from heavy-duty Euro V vehicles induced more toxic effects than Euro VI. Among Euro V vehicles, the exhaust of GTL was the most toxic. The composite weekly sample of the urban background atmosphere showed to be extremely toxic, decreasing cell viability to 2.66%, while the daily road tunnel samples caused a decrease of up to 83.6% at most. In the particulate extracts of the urban background atmosphere, retene and naphthalene were found, which were absent or present at trace levels in the tunnel. Irrespective of the sample, although some changes were observed in the A549 cell cycle after 24 h exposure in all the phases, the differences in the mean values were not great enough to exclude the possibility of random variability. When the results from the three phases (G0/G1, S and G2/M) of the cells exposed to all PM extracts were compared to the control, no statistically significant difference was detected. Regardless of the sample type, no significant increases in cellular ROS levels were observed either.