Inhalation is an important exposure pathway to airborne pollutants such as heavy metals, polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and particulate matter. Chronic exposure to those chemicals, which form part of complex environmental mixtures, may mean important human health risks. In the present study, the suitability of different in vitro tests to evaluate the toxic effects of air PM(10) pollutants is investigated. In addition, it is also assessed how to distinguish the contribution of chemical pollutants to toxicity. Sixty-three air samples were collected in various areas of Catalonia (Spain), and the levels of ecotoxicity, cytotoxicity and genotoxicity were evaluated. Aqueous acidic extractions of quartz fiber filters, where PM(10) had been retained, were performed. The photo-luminescent bacteria Vibrio fischeri (Microtox®) bioassay was performed to assess ecotoxicity. Moreover, MTT and Comet Assays, both using human lung epithelial cells A549 as target cells, were applied to assess the cytotoxicity and genotoxicity of air samples, respectively. The results show that Microtox® is an excellent screening test to perform a first evaluation of air quality, as it presented a significant correlation with chemical contaminants, contrasting with MTT Assay. Although none of the samples exhibited genotoxicity, a high correlation was found between this in vitro test and carcinogenic agents. Urban samples from traffic-impacted areas would be significantly more toxic. Finally, environmental temperature was identified as a key parameter, as higher values of ecotoxicity were found in winter.