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Renewable energy resources like biomass from plant and algae have gained more interest for biodiesel production as an energy source to reduce the consumption of fossil fuels and elevated global warming. In this study, Myxosarcina sp., an unicellular cyanobacterium was evaluated for higher biomass and lipid production by the supplementation of sugar industrial waste (SIW), sodium chloride (NaCl) and media optimization with response surface methodology (RSM) for biodiesel production. The outcome of the findings showed that biomass and lipid productivities of 28.5 ± 2.4 (1.2 fold) and 3.4 ± 0.2 (1.3 fold) mg/L/day were observed in the BG-11 media supplemented with SIW than control (24 ± 1 and 2.6 ± 0.4 mg/L/day). However hyper lipid content 20.6 ± 1.8 % (1.5 fold) was achieved by RSM optimized media including NaCl at 0.8 M, SIW at 2 mL/L, sodium nitrate (NaNO3) at 2.5 g/L and magnesium sulphate (MgSO4) at 0.075 g/L than control (13.6 ± 1.4 %). Fatty acid characterization by GC-MS anlysis revealed that Myxosarcina sp. yielded 39 ± 5.3 % of saturated fatty acids (SFA) and 61 ± 5.3 % of mono unsaturated fatty acid (MUFA) and its biodiesel fuel properties evaluated using empirical equations showed that almost all the properties calculated were in concurrence with the national and international biodiesel standards EN 14214 (Europe), ASTM D6751-02 (US) and IS 15607 (India). Thus, Myxosarcina sp. can be utilized as the environmental friendly biodiesel feedstock for high quality biodiesel production in the current scenario for the escalating energy demand.