The development and use of new technologies for the amplification of the biodiesel production process in the transesterification reaction, which improves the mixing between the two phases, has led to the development and use of new reactors, including micro-reactors. Hence, the present study aimed at investigating the performance of a semi-industrial microreactor in the production of biodiesel from waste cooking oil (WCO) with methanol. The process was carried out through transesterification using acetone as the co-solvent at the presence of limescale (kettle limescale deposit) under different conditions. To optimize the reaction conditions, the main parameters affecting the purity of produced biodiesel, including residence time, reaction temperature, methanol to oil volume ratio, and acetone concentration (oil-based) were studied via response surface methodology (RSM). The best performance in biodiesel production was observed at a residence time of 12.5 min, reaction temperature of 60 °C, methanol to oil volume ratio of 2.15:5, and acetone concentration (pure oil-based) of 13.95 wt%, which resulted in a biodiesel purity of 97.16%. According to the results, the utilization of kettle limescale deposits as the catalyst and acetone as the co-solvent can help to reduce reaction time and production cost.