Due to the increase in fossil fuel prices, limited reserves and environmental damage, researchers turned to renewable energy sources. Studies have been conducted on the use of biodiesel to reduce the use of fossil fuels used in vehicles. In this study, biodiesel obtained from cottonseed oil by transesterification method was used as fuel. Cotton oil methyl ester and methanol, different proportions of fuels (diesel fuel (DF), 20% biodiesel (BD20) by volume, 20% methanol (AL20) and derivatives (BD20AL20) were obtained. The effects of DF and other fuels on the combustion and performance characteristics were discussed. The tests were conducted on a single-cylinder diesel engine at the varying engine speeds from 1750 to 3250 rpm with the intervals of 500 rpm. In comparison with that of conventional DF, the BSFC value of BD20, BD20AL20, and AL20 test fuels has exhibited a significant increment with 10.4%, 39%, and 24%, respectively. DF has the highest value with 28.93% BTE. The reason of the high BTE for DF is the heating value, and high-energy content thereof. In comparison with that of DF, the BTE value of BD20, BD20AL20, and AL20 test fuels has exhibited a significant decrease with 6.8%, 16.74% and 9.5%, respectively. Maximum in-cylinder pressure and maximum heat release rate values are observed for the test fuels containing biofuels due to their low cetane number as compared to that of DF.