Microalgae are considered as one of the most promising feedstocks for biofuel production because of their environmental and social benefits. However, challenges exist in converting microalgal lipids into algal biofuels because of the unique characteristics of microalgae and the technologies for processing them. This study aims at exploring an alternative sub-/supercritical methanol (subCM/SCM) process technology that combines lipid extraction from whole microalgae and lipid esterification/transesterification in a single step or in situ transesterification. A high lipid content microalgal strain, Schizochytrium limacinum, was used for in situ transesterification in a batch reactor. Temperature (170, 210, 250, and 290 degrees C), reaction time (30, 60, 90, and 120 min), and lipid/methanol molar ratio (sRatio; 1:50, 1:75, and 1:100) were investigated for their effects on the conversion efficiency. The temperature appeared as a most positive influential factor. Additionally, the operating temperature over 250 degrees C caused degradation of the lipids and/or algal biomass and, thus, led to the decline of the ester yield. The combination of the reaction time and temperature had a significant impact on the in situ transesterification reaction. The sRatio had a statistically significant impact on the product yield and purity, and both these two response factors reached the maximum levels after the sRatio reached 1:75. It was observed that the highest product purity (37.5 wt %) occurred at sRatio of 1:75, 211.6 degrees C, and 120 min, with a product yield of 58.4 mol %. This study shows that the in situ transesterification of microalgae bears some advantages over the traditional two-step processes and has the potential to be applied to large-scale processing for biodiesel production.