Biodiesel engines produce several intermediate species, which can potentially harm the human health. The concentration of these species and their health risk potential varies depending on engine technology, fuel, and engine operating condition. In this study, experiments were performed on a large number of engines having different configurations (emissions norms/fuel used), which were operated at part load/full load using B20 (20% v/v biodiesel blended with mineral diesel) and mineral diesel. Experiments included measurement of gaseous emissions, and physical, chemical, and biological characterization of exhaust particulate matter (PM). Chemical characterization of PM was carried out for detecting polycyclic aromatic hydrocarbons (PAH's) and PM bound trace metals. The biological toxicity associated with PM was assessed using human embryonic kidney 293T cells (HEK 293T). The mutagenic potential of the PM was tested at three different concentrations (500, 100, and 50 μg/mL) using two different  Salmonella strains, TA98 and TA100, with and without liver S9 metabolic enzyme fraction. PM samples exhibited cytotoxic effect on HEK 293T cells (IC50 < 100 μg/mL) and there was significant potential for reactive oxygen species (ROS) generation. Comparison of different engines showed that modern engines (Euro-III and Euro-IV compliant) produced relatively cleaner exhaust compared to older engines (Euro-II compliant). Biodiesel-fueled engines emitted lower number of particles compared to diesel-fueled engines. However, chemical characterization revealed that biodiesel-fueled engines exhaust PM contained several harmful PAHs and trace metals, which affected the biological activity of these PM, as reflected in the biological investigations. Mutagenicity and cytotoxicity of PM from biodiesel-fueled engines were relatively higher compared to their diesel counterparts, indicating the need for exhaust gas after-treatment.