In the present study biodiesel was produced by various fungal species isolated from Egypt using sugarcane molasses as substrate. In the first stage 6 oleaginous fungi, namely, Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, Fusarium oxysporum, Aspergillus parasiticus and Emericella nidulans var. lata were used for lipid production. Subsequent to fungal cultivation on sugarcane molasses the cultures were filtered and biodiesel was prepared by direct esterification of dry fungal biomass. Methyl esters of palmitic, stearic, linoleic and elaidic represented the major components while palmitoleic represented a minor component of biodiesel produced from tested oleaginous fungi. In the second stage, the spent medium of fungal culture was used as the fermentation medium for hydrogen production by Clostridium acetobutylicum ATCC 824. The maximum total H2 yield was obtained with the spent medium of Epicoccum nigrum and Alternaria alternata. The results presented in this study suggest a possibility of interlinking the biodiesel production technology by fungi with hydrogen production by Clostridium acetobutylicum ATCC 824 to exploit the residual sugars in the spent media and therefore increase the economic feasibility of the biofuel production from molasses