This thesis is built on evaluating efficiencies of internal combustion engines fueled with bio-fuels. The ways of incorporating bio-fuels into practice are described in the theoretical section of this thesis, and are divided in categories. Physical and chemical properties of selected bio-fuels are described as well as manufacturing processes. There is also a view on current legislation dealing with bio-fuels in Czech Republic and European Union. Thesis offers an energy losses and effects on selected efficiencies are described. Thesis also includes description of engine operating parameters and their combustion cycles. Practical part of diploma thesis is dealing with measuring and evaluating operating parameters of four chosen automobiles fueled with bio-fuels. Measured data are in tables, statistics and graphics are commented and part of thesis. Based on evaluation of operating parameters it was figured out that effective efficiency increased for all measured cases while running on bio-fuels.