Particulate matter arising from different sources affects air quality, representing a risk for human health and the environment. The Valle de Aburrá, Colombia, is facing serious air pollution conditions due to its geographical location and the continuous vehicle fleet growth. In this study, the chemical composition and morphology of the PM10 (particulate matter with aerodynamic diameter < 10 μm) collected at monitoring stations across the Valle de Aburrá, were studied by using thermogravimetric analysis (TGA), elemental analysis, scanning electron microscope/energy-dispersive X-ray spectrometer (SEM/EDS), and gas chromatography-mass spectrometry (GC–MS). Additionally, the potential of PM10-induced cytotoxic and oxidative effects was evaluated using peripheral blood mononuclear cells (PBMCs). The PM10 from Valle de Aburrá has a chain morphology and particles of irregular shape; it is mainly composed of ashes (54.8%), followed by volatiles and fixed carbon. Furthermore, different metals were found in PM10, including Si, Fe, K, Na, Al, Cr, and Pt. On the other hand, the polycyclic aromatic hydrocarbon (PAH) analysis showed benz[a]anthracene levels at 6.71 ng cm−2 which may act as a potent carcinogen by generating various reactive metabolic intermediates leading to oxidative stress. These results are consistent with the cytotoxic effect and the production of ROS observed in PBMCs. Finally, the results suggest that the PM10 of the Valle de Aburrá is mainly arising from construction waste transport, coal combustion to generate energy, and vehicles, representing a potential health risk for the citizens.