In the current work, carotenoids of the three microalgae [Spirulina platensis (S. platensis), Scenedesmus obliquus (S. obliquus) and Dunaliella salina (D. salina )] were extracted, quantified and exposed to phytochemical and biological (cytotoxic, antiviral, antioxidant, and anticholinesterase) activities . Also, some stress conditions were applied to S. platensis and S. obliquus to raise their carotenoid content. D. salina was found to contain the highest percent of carotenoids (0.67%) followed by S. obliquus (0.64 %), then S. platensis (0.5%). Six carotenoids were detected and identified in S. platensis and S. obliquus while four carotenoids were detected in D. salina. Lutein, β -carotene and diatoxanthin were predominant in S. platensis while β -carotene, diatoxanthin and echinenone were predominant in D. salina. Furthermore, echinenone, diatoxanthin and anthraxanthin were predominant in S. obliquus. D. salina carotenoid fraction showed potent cytotoxic activity on hepatocellular carcinoma (HepG2), breast cancer (MCF-7) and colorectal cancer (HCT116) cell lines with an IC50 24.7, 66.4 and 46.7 µg/ml, respectively. Also, S. platensis carotenoid fraction showed a significant cytotoxic activity on HepG2, MCF7 and HCT116 cell lines with an IC50 31.1, 69.9 and 62 µg/ml, respectively. D. salina and S. platensis exhibited significant antioxidant activity using1, 1- diphenyl-2picryl-hydrazyl (DPPH) in a concentration dependent manner. Also, D. salina and S. obliquus inhibited the activity of acetylcholinesterase to 60.00 and 30.00% at 10 µg/ml dose, respectively. So, D. salina and S. platensis carotenoid fractions are recommended to be included in anticancer drug researches while D. salina and S. obliquus carotenoid fractions should have a consideration in the treatment of neuro- degeneration disease.