The use of alternative fuels or biofuel blends could be a way to reduce the environmental burden of increasing traffic. The aim of this study was to compare emissions from conventional fuels and alternative biofuels for diesel and spark-ignition engines under comparable conditions, i.e., using the World Harmonized Transient Cycle for a heavy-duty diesel engine and the Artemis CADC driving cycle for automobiles powered by gasoline and alternative fuels. Total contents of Ba, Ce, Cd, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn were determined in emissions, fuels, and lubricating oils. In addition, the bioaccessibility of metals in emissions was also assessed by extraction in water and in simulated lung fluid (Gamble's solution). Total particulate mass emissions, expressed per kilogram of fuel, and total contents of metals were higher for the diesel engine than for spark-ignition engines. The highest metal contents in emissions from diesel and gasoline fuels were found for Fe, Zn, and Cu. Fe and Cu in emissions from diesel and spark-ignition engines declined with the addition of bio-components in fuels. However, there was no significant decrease in the contents of other metals in emissions from biofuels. The highest degrees of bioaccessibility were observed for Ba, Zn, Cd, and V in emissions from diesel and biodiesel (according to their solubility in water). On the basis of this study, the use of biodiesel (especially methylesters of rapeseed oil) can be recommended to reduce the total mass of particulate and metal emissions from diesel engines.