Transesterification process involves different immiscible substrates includes feedstock oil, alcohol and catalyst to produce fatty acid methyl esters, i.e. biodiesel. However, a poor mixing of the substrates in the transesterification process could lead to a low triglyceride conversion into biodiesel. Appropriate design for mixing is important to promote better surface contact between the substrate to enhance the transesterification reaction. In addition, mechanical assisted mixing system in the intensified process with ultrasonic could help to improve the conversion percentage. In this study, the effect of high frequency ultrasonic in enzymatic transesterification process was investigated. The biodiesel conversion percentage for the enzymatic transesterification of palm oil with methanol via 3 different mechanical mixing systems, i.e., conventional shaking method, high frequency ultrasonic method with and without mechanical stirring, were evaluated. The transesterification reaction was carried out for 12 h at 35 °C with an alcohol-to-oil molar ratio of 6:1, a 10 wt% of liquid enzyme solution, with addition of 16 wt% of water (relative to the weight of enzyme). The results from this study indicated that an enhancement of biodiesel conversion percentage was achieved via high frequency ultrasonic assisted enzymatic transesterification compared to conventional shaking method. The biodiesel conversion percentage obtained via conventional shaking method and high frequency ultrasonic-assisted transesterification processes were 29% and 66%. Meanwhile, the application of additional mechanical stirring into high frequency ultrasonic process further increased the conversion percentage from 66% to 89%. The findings for this study bring a better understanding in high frequency ultrasound assisted enzymatic transesterification. There is a potential reducing the reaction time for enzymatic transesterification with high frequency ultrasound assisted process. This could provide an alternative transesterification process with milder operating conditions.