Biodiesel is an alternative fuel with growing usage in the transportation sector. To compare biodiesel and petroleum diesel effects on particle emissions, engine dynamometer tests were performed on a Euro II engine with three test fuels: petroleum diesel (D), biodiesel made from soy bean oil (BS) and biodiesel made from waste cooking oil (BW). PM2.5 samples were collected on Teflon and quartz filters with a Model 130 High-Flow Impactor (MSP Corp). Organic (OC) and elemental (EC) carbon fractions of PM2.5 were quantified by a thermal-optical reflectance analysis method and particle size distributions were measured with an electrical low pressure impactor (ELPI). In addition, the gaseous pollutants were measured by an AMA4000 (AVL Corp). The biodiesels were found to produce 19–37% less and 23–133% more PM2.5 compared to the petroleum diesel at higher and lower engine loads respectively. On the basis of the carbon analysis results, the biodiesel application increased the PM2.5 OC emissions by 12–190% and decreased the PM2.5 EC emissions by 53–80%, depending on the fuel and engine operation parameters. Therefore OC/EC was increased by three to eight times with biodiesel application. The geometrical mean diameter of particles from biodiesels and petroleum diesel had consistent trends with load and speed transition. In all the conditions, there is a shift of the particles towards smaller geometric mean diameter for the biodiesel made from waste oil.