The main purpose of this study is to provide information on the biochemical composition of the total lipid extract of three different native freshwater microalgae namely Acutodesmus obliquus CN01, Chlorella sp. Carolina-15-2069, and Chlorella vulgaris NIES-1269 at different growth phases, cultured under standardized conditions that will be useful for commercialization purposes due to the content of a-linolenic acid (ALA), a type of omega-3 fatty acids. We monitored growth, biomass accumulation, chlorophyll content, lipid content and fatty acids composition in all three freshwater microalgae. In addition, this study seeks to throw some light on the lipid content of a newly isolated freshwater green alga Acutodesmus obliquus CN01 which have shown to give significant quantities of ALA, an added nutritional value to this microalga oil. Acutodesmus obliquus CN01 exhibited the fastest growth rate among the three microalgae used. Biomass was shown to be enhanced in the nitrogen enrichment medium. The outcomes from this study indicated that newly isolated Acutodesmus obliquus CN01 from Malaysia cultured under normal condition showed fast growth rate with high lipid content and also high ALA content. Therefore, from this study, Acutodesmus obliquus CN01 was identified as species of choice to be further explored for its potential to produce omega-3 fatty acids.