In this study, two freshwater cyanobacteria, Oscillatoria sp. 50A and Synechocystis sp. NN have been evaluated for biodiesel production. Among the two cyanobacteria, Synechocystis sp. NN was isolated, identified by its 16S rRNA gene sequencing. Effects of sodium bicarbonate (SBC), tannery effluent (TE), coir pith (CP) and light stress (L1) on biomass and lipid production of Synechocystis sp. NN were studied. Result showed that maximum biomass productivity of 18.7 ± 0.9 mg/L/day (1.9 folds) was observed in TE supplemented BG-11 media than normal BG-11 media. Meantime, maximum lipid productivity of 2.6 ± 0.4 mg/L/day (1.4 folds) was observed in CP supplemented BG-11 media than normal media. Further, fatty acid composition analyses by GC–MS showed that C16, C18:1 in Oscillatoria sp. 50A and C16, C20:1, C22:1 in Synechocystis sp. NN were predominant and the fuel properties were also in accordance with the international standards. Besides gene expression of acetyl CoA carboxylase D of Synechocystis sp. NN, analyzed by RT-PCR revealed that transcripts of accD were up-regulated by 1.2–4.7 folds in different media conditions. The findings of this study showed that Synechocystis sp. NN can be utilized as a suitable feedstock that is amenable for cultivation using wastes as nutrient source.