It is important to reduce the negative environmental effects of non-road diesel engines, which are increasingly used in many facilities and machines, without loss of performance. Biodiesel is used as an alternative to fossil-based diesel fuels to eliminate these effects and ensure sustainability in energy. This study focused on the optimization of the operating parameters of a non-road diesel engine operating with a waste frying oil biodiesel mixture at 50% load. Pure biodiesel, 1-heptanol, different injection advances and pressures were determined as input parameters for optimization. The tests were designed according to Taguchi’s L16 orthogonal array. ANOVA analysis was performed to determine the importance of input parameters on engine performance and exhaust emissions. Optimization was made based on the highest brake thermal efficiency (BTE) in addition to the lowest values of brake-specific fuel consumption (BSFC), brake-specific hydrocarbon (BSHC), brake-specific nitrogen oxide (BSNOx) and smoke emissions. In the optimization carried out according to the response surface methodology (RSM), the optimum combinations to obtain the best engine characteristics were determined as 17.27% 1-heptanol, a 226-bar injection pressure, 27 CAD injection advance and B75. These optimization results were verified by engine experiments within the recommended error range.