In this study oleaginous yeast, Rhodosporidium kratochvilovae HIMPA1 grown in glucose synthetic medium containing different nitrogen (1 g/l and 0.1 g/l) and phosphorus (0.05 g/l and 0.1 g/l) limited conditions. Among various N and P-limited conditions, the highest lipid content (60.34± 0.69%) obtained under synchronized limitation of N and P (0.1 g/l N and 0.05 g/l P). Live fluorescent cell imaging of yeast cells after BODIPY505-515 nm staining endorses the results of triacylglycerol (TAG) accumulation in lipid droplets. The cells grown in synchronized limitation of N and P exhibited boosted cell size (6.76 ± 0.39 μm) and lipid droplet size (5.62 ± 0.28 μm). Under synchronized limitation of N and P, supersized irregular shaped lipid droplets (LD) coalesced to form big lobules in the cellular compartment of oleaginous yeast having 87.14% enhanced TAG accumulation as depicted by TLC. Synchronized nutrient limitation diverts the CDP-DG pathway of phospholipids synthesis towards de novo TAG synthesis. The maximum increment of oleic acid (C18:1) was reported in synchronized limitation of N and P that improve the biodiesel properties like oxidative stability, viscosity, cetane number and cold filter plugging point.