Being a renewable fuel, biogas can be produced either through anaerobic digestion from animal manure, inedible seed residue, food waste, agricultural waste, domestic waste and sewage sludge or through thermo-chemical processes. Along with thermal applications, by means of combustion inside an engine, biogas can produce both mechanical and electric energy. Depending on the inertia forces of the active parts and the characteristics of the in-cylinder combustion of the engine, vibration can be quite a big problem because it leads to rapid breakdown of engine parts, noisy operation and decreased performance and power output. The CO2 in biogas is an important compound that affects its lower heating value (LHV), burning characteristics and exhaust emissions. There are a limited number of studies in the literature on the effect of biogas CO2 content on engine vibration. In the present study, a four-cylinder diesel engine was modified to operate with a spark plug and used to detect the effect of the biogas CO2 content on the vibration of the engine. The experiments were carried out with biogas containing 13% and 49% CO2 at 1.5-9 kW with 1.5 kW load increments at a steady 1500 rpm speed. The amplitude of the engine vibration at all three axes increased as the CO2 ratio in the biogas and the engine load increased. On the other hand, by decreasing the CO2 ratio and increasing the engine load, cylinder pressure increased and brake specific fuel consumption decreased. The highest amplitude of engine vibration for all loads was observed at the lateral axis.