Biodiesel is a renewable fuel conventionally prepared by transesterification of pre-extracted vegetable oils and animal fats of all resources with methanol, catalyzed by strong acids or bases. This paper reports on a novel biodiesel production method that features acid-promoted direct methanolysis of cellular biomass of oleaginous yeasts and filamentous fungi. The process was optimized for tuning operation parameters, such as methanol dosage, catalyst concentration, reaction temperature and time. Up to 98% yield was reached with reaction conditions of 70 °C, under ambient pressure for 20 h and a dried biomass to methanol ratio 1:20 (w/v) catalyzed by either 0.2 mol L−1 H2SO4 or 0.4 mol L−1 HCl. Cetane numbers for these products were estimated to range from 56 to 59. This integrated method is thus effective and technically attractive, as dried microbial biomass as feedstocks omits otherwise tedious and time-consuming oil extraction processes. Copyright © 2007 Society of Chemical Industry