Dual application of biomass for biohydrogen and biodiesel production could be considered a feasible option for economic and sustainable energy production from microalgae. In this study, after a large screening of fresh water microalgal isolates, Scenedesmus sp. NBRI012 and Chlorella sp. NBRIO29 have exhibited high biomass (1.31 +/- 0.11 and 2.62 +/- 0.13 g/L respectively) and lipid (244.44 +/- 12.3 and 587.38 +/- 20.2 mg/L respectively) yield with an organic carbon (acetate) source. Scenedesmus sp. NBRI012 has shown the highest H-2 (maximum evolution of 17.72% v/v H-2 of total gases) production; it produced H-2 continuously for seven days in sulfur-deprived TAP media. Sulfur deprivation during the H-2 production was found to increase the lipid content (410.03 +/- 18.5 mg/L) of the residual biomass. Fatty acid profile of the lipid extracted from the residual biomass of Scenedesmus sp. NBRI012 has showed abundance of fatty acids with a carbon chain length of C16 and C18. Cetane number, iodine value, and saponification value of biodiesel were found suitable according to the range given by the Indian standard (IS 15607), Brazilian National Petroleum Agency (ANP255) and the European biodiesel standard EN14214. (c) 2015 Elsevier Ltd. All rights reserved.