ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are harmful compounds because of their toxicity, adverse effects on human health, and threats to engine components (i.e. wetstacking). With the increased use of renewable fuels, it is imperative that the formation of PAHs be examined, in detail, with respect to the combustion characteristics and aromatic content of biofuels used in diesel engines. Since biodiesel is free of aromatic content in its chemical structure, this study provides comparative assessments of the PAHs, toxicity and regulated emissions of biodiesel (B100) and diesel fuel (D100), based on the fuel aromaticity and engine characteristics when used in two diverse engines. Results indicate that the use of B100 in both engines reduced total PAH emissions by 48.02% − 49.36% and PAH toxicity by 83.49% − 84.8% as compared to diesel fuel, which is a clear indication of the importance of the aromatic content of the base fuel on PAH formation and toxicity (i.e. aromatic-free fuel produces less PAH than diesel with high aromaticity). The aromatic ring distribution associated with B100 exhaust consisted of 50% two, 34% three and 16% four aromatic rings for the Onan engine, and 53% two, 32% three and 15% four aromatic rings for the Kubota engine. Benzo[a]pyrene, a five-ring PAH with great carcinogenic threat, was eliminated in both engines as a result of using B100. To demonstrate the significance of combustion processes on PAH formation, performances of the engines were compared and it was determined that the Kubota engine performed better for both fuels in reducing PAH emissions, due to such features as its injection system and combustion chamber design as compared to the Onan engine. Therefore, it has been concluded that PAH emissions and toxicity strongly depend on both the aromatic content and the combustion processes, as aromatic-free biodiesel significantly decreased PAHs along with additional reduction in PAHs and toxicity depending on the structural specifications of the diesel engine. Based on this study, aromaticity has been determined to be more effective than the type of engine in use for PAH reduction.