Organic and water extracts of Isochrysis galbana T-ISO (=Tisochrysis lutea), Tetraselmis sp. and Scenedesmus sp. were evaluated for their antioxidant activity, acetylcholinesterase (AChE) inhibition, cytotoxicity against tumour cell lines, and fatty acids and total phenolic content (TPC). I. galbana T-ISO had the highest TPC (3.18 mg GAE g−1) and radical scavenging activity, with an IC50 value of 1.9 mg mL−1 on the acetone extract. The extracts exhibited a higher ability to chelate Fe2+ than Cu2+, and the maximum Fe2+ chelating capacity was observed in the hexane extract of Scenedesmus sp. (IC50=0.73 mg mL−1) and Scenedesmus sp. (IC50 = 0.73 mg mL−1). The highest ability to inhibit AChE was observed in the water and ether extracts of Scenedesmus sp., with IC50 values of 0.11 and 0.15 mg mL−1, respectively, and in the water extract of I. galbana (IC50 = 0.16 mg mL−1). The acetone extract of I. galbana T-ISO significantly reduced the viability of human hepatic carcinoma HepG2 cells (IC50 = 81.3 μg mL−1) as compared to the non-tumour murine stromal S17 cell line, and displayed a selectivity index of 3.1 at the highest concentration tested (125 μg mL−1). All species presented a highly unsaturated fatty acids profile. Results suggest that these microalgae, particularly I. galbana T-ISO, could be a source of biomolecules for the pharmaceutical industry and the production of functional food ingredients and can be considered as an advantageous alternative to several currently produced microalgae.