Aim: The aim of the study was to isolate and characterize freshwater microalgae from Puzhal lake, Chennai, Tamil Nadu, and to analyze its biochemical composition, poly-unsaturated fatty acid content, anticancer activity, cytotoxicity, and biodiesel properties. Methodology: Pure isolate obtained from freshwater was characterised biochemically and identified by ITS-2 RNA sequence as Monoraphidium griffithii. The microalgal culture was maintained under white light illumination for a photoperiod of 16:8 light/dark hours respectively. The saturated and poly-unsaturated fatty acid profiling was performed in a Gas Chromatography-Flame ionization detector and the biodiesel properties were analyzed through BiodieselAnalyzer© Ver. 2.2 software. The anti-cancer and cytotoxicity studies were performed using the human lung cancer cell line (A549) and Vero cell line at 20, 40, 60, 80, and 100 µg biomass/ml concentration. Results: The biochemical composition was determined to be 32.97%±1.648 protein, 15.36%±2.857 carbohydrate, 15.89%±1.407 lipid, 26.27±1.48 µg/mg chlorophyll a+b and 8.03±1.92 µg/mg carotenoid. The fatty acid profiling revealed the presence of 19 different fatty acids of which Palmitic acid was 18.08%, cis-10 heptadecanoic acid was 17.37%, alpha-linolenic acid was 2.05%, and docosahexaenoic acid was 1.27%. The biodiesel properties were within the American standard for testing materials (ASTM) limits. 46.5% cell viability was attained at a microalgal concentration of 80 µg/ml against the human lung cancer cell line (A549) and the cell viability was 77.87% even at the high concentration of 100 µg/ml against the Vero cell line. Conclusion: The isolated microalgae Monoraphidium griffithii can be used for the production of biodiesel. The presence of alpha-linolenic and docosahexaenoic acid and anticancer activity makes them a source for human and animal nutrition.