The aim of the study was to determine impact of commercially available hydrotreated vegetable oil (HVO) and its mixture (HVO5, where 5% (v/v) HVO and 95% (v/v) FDD) with diesel fuel (FDD) on the power, torque, fuel consumption, and exhaust gas composition of an atmospheric internal combustion diesel engine used in off-road applications. Diesel fuel was used as the comparative fuel. Testing was realized in a full-load mode on the KOHLER KDI 1903 M 3-cylinder diesel engine on a SIERRA CP-Engineering engine test bench. The AVL SESAM FTIR exhaust gas analytical system was used to determine exhaust gas emissions, while the AVL KMA Mobile fuel consumption measuring device was used to measure fuel consumption. Research showed that the lowest power and torque readings were obtained with FDD, while HVO showed a slightly higher result compared to the fossil diesel fuel. At the same time, the highest hourly fuel consumption was observed running on HVO5, while the lowest was observed with FDD. Increases in carbon monoxide (CO), carbon dioxide (CO2), and nitrogen oxide (NOx) emissions were observed for HVO5 compared to those of FDD. The CO content in emissions increased by an average of 3.0% using HVO and by an average of 36% using HVO5, but the NOx content in the emissions increased by an average of 3.0% using HVO and by an average of 8.8% using HVO5. The reduction by an average of 60% using HVO in emissions was found in the case of hydrocarbons (HC). Research confirmed that the physicochemical properties of HVO could leave an impact on the main engine performance parameters and exhaust emissions.