The interest in bioactive compounds from microalgae is increasing since they have medicinal and nutritional areas. The present work aims to evaluate the potential pharmaceutical interest of extracts from three eustigmatophyte strains from the Coimbra Collection of Algae (ACOI): Chlorobotrys gloeothece, Chlorobotrys regularis and Characiopsis aquilonaris. Antioxidant and antiproliferative activities were determined as well as chlorophyll a, carotenoid and phenolic total contents. In addition, major pigments and sterols were identified and quantified. The three strains were grown until the stationary phase and then the biomass was extracted. Antioxidant activity was measured by TEAC, DPPH and FRAP assays and antiproliferative effect was assessed by the MTT method on MCF-7, PC-3 and NHDF cells. The pigment and phenolic total contents were determined by spectrophotometry. Of these strains, C. aquilonaris showed the highest antioxidant activity measured by TEAC and FRAP assays (23.98 +/- 0.01 mu mol TE eq g(-1) DW and 42.57 +/- 0.04 mu mol TE eq g(-1) DW, respectively), a selective effect in reduting MCF-7 cells proliferation and a larger amount of chlorophyll a, carotenoids and phenolic content (18.40 +/- 0.00 mu g chlorophyll a mg(-1) DW, 2.27 +/- 0.00 mg carotenoids g(-1) DW and 6.23 +/- 0.01 mg GAE g(-1) DW, respectively). A positive correlation between chlorophyll a and TEAC assay was observed, as well as between carotenoids and TEAC and FRAP assays, suggesting these compounds as important contributors to significant antioxidant activity. Violaxanthin, cholesterol and stigmasterol were present in larger amount in C. aquilonaris while C. regularis showed a higher amount of beta-carotene. These results suggest that these three ACOI eustigmatophytes are promising for applications in the improvement of human health, particularly in cancer prevention and treatment.