Exploring and enhancement of biodiesel production from feedstock like non-edible vegetable oil is one of the powerful method to resolve inadequate amount of conventional raw materials and their high prices. The main aim of this study is to optimize the biodiesel production process parameters of a biodiesel obtained from non-edible feedstocks, namely Neem (Azadirachta indica) oil and Sesame (Sesamum indicum L.) oil, with response surface methodology using Doehlert’s experimental design. Based on the results, the optimum operating parameters for transesterification of the mixture A50S50 oil mixture at 51.045° C over a period of 45 minutes are as follows: methanol-to-oil ratio: 8.45, and catalyst concentration: 1.933 wt.%. These optimum operating parameters give the highest yield for the A50S50 biodiesel with a value of 95.24%. The results from the analysis show that there is a substantial improvement in the physicochemical properties of the A50S50 biodiesel after optimization, whereby the kinematic viscosity at 40° C, density at 15° C, calorific value, acid value and oxidation stability is 4.250 mm2 /s, 835.2 kg/m3, 40.62 MJ/kg, 0.032 mg KOH/g and 9.21 h, respectively. The physicochemical properties of the optimized A50S50 biodiesel satisfied the requirements given in the ASTM D6751 and EN14214 standards.