Atmospheric particulate matter (PM) characterizes the atmospheric air quality. PM particles can adsorb and include several toxic air pollutants of urban areas. The current study aimed to develop an experimental procedure to assess the toxicity of the pollutants on PPM10 by means of the comet assay on earthworms directly exposed to PM10 collecting filters. A particular focus was the amount of polycyclic aromatic hydrocarbons (PAHs) in the filter, in spite of their very low concentration in PM, because of their strong mutagenic and carcinogenic effects. A quartz filter exposed to polluted air containing 24.9 mg/g of PM10 and 14.1 μg/g of PAHs was characterized and mechanically reduced into a very fine powder by means of a planetary ball mill. This powder was combined with artificial soil samples allowing treatments at 15 μg/g of PM10 (0.008 μg/g of PAHs), 22.5 μg/g of PM10 (0.012 μg/g of PAHs), 30 μg/g of PM10 (0.016 μg/g of PAHs). Earthworms were exposed to each treatment for seven days, including blank treatments with powdered clean quartz filter, such as phenanthrene (used as the standard), and an untreated soil. DNA damage was observed starting from 0.012 μg/g of PAHs in 22.5 μg/g of PM10. No single PAH was detected or quantified in the bodies of the earthworms after microwave assisted solvent extraction (MASE) and GC-MS analysis. The results demonstrate that even a very low amount of PM10 absorbed by the earthworms had a toxic effect on their immune systems, which could also have been caused by other xenobiotics included into the filter.