Coniochaeta hoffmannii was isolated from soils contaminated with biscuit factory wastes showed the maximum lipid accumulation capacity in the study. Lipid production was optimized in terms of pH, carrot pomace loading, nitrogen type and amount, incubation time. Solvent, alcohol type and catalyst concentration, dried/wet biomass concentration, reaction approaches and time were optimized for lipid extraction and transesterification. The highest lipid accumulation was found as 52.0% at pH 4 in the presence of 10% carrot pomace, 0.5 g/L cheese whey at the end of the 48 h incubation. The maximum total C16 and C18 FAME rates were detected at the 25 °C, in the presence of 4 g/L dried C. hoffmannii biomass, methanol and 3% NaOH by using the in-situ transesterification process at the end of the 0.5 h as 96.3%. This is the first report about the usage of C. hoffmannii lipids obtained from carrot pomace for sustainable biodiesel production.