The conversion parameters in biodiesel production have a substantial impact on the yield of methyl esters, consequently affecting the physicochemical properties of the fuel. Tucuma and Ungurahui bio-oils were used in this study to optimize the process parameters. A total of 27 transesterification experiments were conducted for each bio-oil, and the results were statistically investigated using the RSM approach. In addition, advanced kinetic modelling was performed using Aspen Plus software tools on the Tucuma biodiesel transesterification process to investigate the key effect of process parameters on the fuel properties. The study found a greater yield of 99.4 % and 99.5 % for Tucuma and Ungurahui, respectively, under optimized parametric conditions. ANOVA tests revealed lower p-values for catalyst and temperature, indicating their significant influence on the conversion process. For both fuels, GC–MS tests recorded 100 % methyl esters, and fuel properties agree with the ASTM biodiesel standards. Notably, the viscosity of both biodiesels is 4.0 mm2/s, which closely matches that of diesel fuel. Furthermore, the process parameters such as methanol-to-oil ratio, catalyst, and temperatures influenced the methyl esters such as oleate, linoleate, and palmitate, impacting properties density and calorific value. Besides, viscosity was affected by the time parameter, which in turn influenced methyl stearate. The study concludes that while the process parameters influenced the fuel properties, their overall impact on the change was marginal.