Road transport is increasing all around the globe and biofuels have come to the forefront of public interest. According to Article 3, Directive 2009/28/EC, each member state has to ensure that an energy share from renewable sources in all forms of transportation reaches at least 10% of the final consumption of energy in transportation until 2020. The blending of biofuels is one of the methods available to member states to meet this target and it might even be expected to be a main contributor. This article analyses and compares selected biofuels, their chemical properties and their influence on engine operational parameters. The operational parameters of the diesel engine of the Skoda Roomster 1.4 TDI were measured on a chassis dynamometer according to the NEDC driving cycle, and pure diesel fuel, HVO and a blend of fuels (diesel fuel, HVO and butanol) were used for comparison. Operation on biofuels shows a slight decrease in performance parameters up to 10% and an increase in emission production (especially CO in the case of D50H30B20). Positive influences of biofuels were proven with a decrease in exhaust gas opacity and particulate matter production, up to 50% in the case of D50H30B20.