Green microalga Monoraphidium sp. was isolated during the mass cultivation study of Ettlia sp., when it was found that the culture was dominated by smaller unidentified cells with high lipid content. The strain was identified and named as Monoraphidiumsp. based on itsmorphological characterization under light and confocal microscopy followed by genomic characterization via 18 s sequencing. The lipid content, fatty acid profiles, biomass and lipid productivity of microalga were examined under autotrophic cultivation condition using various CO2 concentrations as well as different nitrogen sources. Isolated Monoraphidium sp. was found to have high lipid content of 28.92% determined via gas chromatography and sulfo-phospho-vanillin method (SPV). Lipid profile of the algal cells exhibited a suitable distribution of fatty acids for biodiesel production, with cultivation using urea producing particularly high quality of biodiesel. Therefore, the results suggest that Monoraphidium sp. is a promising feedstock for biodiesel production.