There are two main reasons of alternative fuel search of scientists: environmental problems resulted from combustion of fossil fuels and limited reserves of crude oil. Biodiesel and Hydrogen (H-2) are two of the most promising alternative fuels with their environmental friendly combustion profiles. The aim of this study was to evaluate vibration level of a hydroxyl (HHO) gas generator installed and diesel engine using different kinds of biodiesel fuels. In this study, at different flow rates, the effect of HHO gas addition on engine vibration performance was investigated with a Mitsubishi Canter 4D34 -2A diesel engine. HHO gas introduced to the test engine via its intake manifold with 2, 4 and 6 L per minute (LPM) flow rates when the engine was fuelled with sunflower, canola, and corn biodiesels. The vibration data was collected between 1200 and 2400 rpm engine speeds by 300 rpm intervals. Finally, artificial neural network (ANN) approach was conducted in order to predict the effect of fuel properties and HHO amount on engine vibration level. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.