One of the challenging tasks for the researchers is to identify alternative renewable energy feedstocks which can be used as a substitute for fossil fuel to meet the increasing demand for energy, reduce the depletion of fossil fuel reserves and eliminate the increasing concerns of environmental impact. The 2nd generation poppy (Papaver somniferum) seed oil (PSO) can be considered as one of the feedstocks which would be low cost and does not compete with food crops for biodiesel production. In this study, biodiesel production was conducted in 2 stage processes which consist of acid and base–catalysed esterification and transesterification to reduce the free fatty acid (FFA) content of poppy seed oil (PSO) and to increase the yield of poppy seed oil methyl ester (PSOME), respectively. To determine the maximum yield of methyl ester, different parameters that influence the production of methyl ester, namely the molar ratio of methanol to oil (5.0:1–7.0:1), catalyst loading (0.5–1.5 wt.%), reaction temperature (40–80 °C) and time (60–120 min) were investigated under the transesterification reaction. The results found that methanol to oil molar ratio of 6.0:1, catalyst loading of 1.0 (wt.%), reaction temperature of 60 °C and reaction time of 90 min were observed to be the optimum process parameters for maximising the yield of PSOME at a constant speed of 720 rpm. In these conditions, the maximum methyl ester yield was found to be about 93 %. The gas chromatography (GC) analysis was conducted to evaluate the fatty acid methyl ester (FAME) composition of the PSOME. Furthermore, the physico-chemical properties of the PSOME were evaluated and characterised using ASTM and EN standards for biodiesel. It was found that PSO can be used as a potential feedstock for biodiesel production as most of the physico-chemical properties are within the ASTM and EN standards.