The need to substitute fossil fuels with energy alternatives has favored the development of biodiesel and green diesel, which generate less amount of particulate matter (PM), but their biological reactivity is unknown, an important factor of quality and risk. Therefore, the genotoxicity and mutagenicity potential of PM emitted by the combustion of mixtures of green diesel and palm biodiesel with petroleum diesel has been evaluated; 10% palm biodiesel (B10), 10% and 30% green diesel (HVO10 and HVO30) and ultralow sulfur diesel (ULSD) were subjected to combustion at two engine operating speeds: 1890 and 2410 rpm. From each PM sample, the organic fraction was extracted to evaluate the mutagenicity with the Ames test in the presence and absence of microsomal enzymes, and the genotoxicity with the alkaline comet assay, while in the Ames test, concentrations of equivalent 3, 1.5, and 0.8 μg of PM/µl were considered; in the comet assay, equivalent 3.1, 6.3, and 12.5 μg of PM/µl were tested. The organic fraction of the PM from the ULSD was the least genotoxic and mutagenic. In contrast, the organic fraction of the PM of HVO30 showed the highest genotoxic and mutagenic activity (p < 0.05). Although the genotoxic potential of PM generated at 1890 rpm, low speed, was slightly higher, no relationship was found regarding the engine operating speed. The results of this investigation show that the fuel extraction method and the mixture with the petroleum diesel affect the quality of the PM and, therefore, its biological activity. Consequently, the physicochemical monitoring of the PM should be complemented with biological procedures to evaluate the quality of alternative fuels and protect the environmental health.