Abstract: In recent decades, microalgae have acquired attention from pharmaceuticals to biofuels. The growth and total chorophyll content of three economically important microalgae (Chlorella sp. NTAI01, Monoraphidium sp. NTAI02 and Scenedesmus sp. NTAI03) isolated from fresh water body in five selected culture media on different days of incubation was studied for biomass production. Biomass feedstock has reviewed great interest to be used as an alternative and renewable source of energy. All the three organisms showed varied growth pattern and total chlorophyll content in different culture media. However the growth and total chlorophyll content of Chlorella sp. NTAI01 and Monoraphidium sp. NTAI02 was optimum in Half strength Chu 10 medium. In case of Scenedesmus sp. NTAI03 the growth and total chlorophyll content was found to be significant in Bold’s Basal medium. The Acidified Bold’s Basal medium and BG-11 medium fairly supports the growth of all the three microalgae whereas the Modified Hoagland’s medium does not support the microalgal growth. The optimized growth medium will be used for biomass production for biofuel application.