Currently, aquaculture sector is one of the most significant and rapidly expanding industries in global food production. Ensuring both optimal nutritional content and sustainability of ingredients within commercial aquaculture feed represents primary challenges in meeting the dietary needs of farmed aquatic species. The utilization of preferred fish meal and fish oil can exacerbate sustainability issues attributed to the risk of overfishing and environmental degradation. Hence, aquaculture sector is actively pursuing sustainable alternatives to surrogate fish meal and fish oil, recognizing their crucial nutritional role for farmed aquatic species. Microalgae, particularly those rich in fucoxanthin and lipids, hold promising potential as nutritional supplements for aquaculture feed. Furthermore, Malaysia is a tropical country with megadiverse ecosystems that provides a rich landscape for bioprospecting endeavors, unlocking the vast potential of microalgae for innovative applications in various industries. These indigenous microalgae could be cultivated superior under particular local environmental conditional and meanwhile maintains th e balance of native ecosystems. Therefore, this study aimed to bioprospect indigenous freshwater and marine species from Malaysia, focusing on their fucoxanthin and lipids contents for aquaculture purpose. A total of 11 microalgae strains (six freshwater and five marine microalgae) were included in this study. The growth properties and fucoxanthin content of these microalgae were initially evaluated. Then, three of the highest fucoxanthin -producing microalgae from freshwater and marine environments, respectively, were selected for lipid and fatty acid methyl esther analysis. Chaetoceros calcitrans (UPMC-A0010) (marine microalgae) produced the greatest amount of fucoxanthin (11.40 ± 0.38 mg/g DW) and eicosapentaenoic acid (EPA) (13.05 ± 0.23 % total fatty acids (TFA)) than the investigated microalgae. Additionally, one freshwater microalga, Mayamaea permitits (UPMC-A0071) demonstrated substantial amount of fucoxanthin (8.36 ± 1.23 mg/g DW) and EPA (7.25 ± 0.18 % TFA). This work establishes a crucial foundation for the advancing microalgal research and bolstering the economic feasibility of producing these valuable compounds. By leveraging these two Malaysian indigenous microalgae, this study paves the way for their potential application in the aquaculture sector.