Microalgae have attracted interest as a potential feedstock for biodiesel and valuable co-products such as bioethanol and protein. However, production at a commercial scale is still costly in part due to a lack of knowledge of their biology which in turn limits our ability to increase their overall growth rate or alter their metabolism and composition through changes in culture conditions or genetic engineering. We therefore isolated ten strains of freshwater microalgae (Chlorophyta) and characterized them at the morphological, molecular and biochemical level under autotrophic conditions. Two Scenedesmus strains (BR003 and BR024) differed from others strains in respect to their fatty acid profile, with high levels of C16 and C18:1 and low levels of polyunsaturated fatty acids. Interestingly, Chlamydomonas sp. BR020 and Chlorella vulgaris BR017 showed high levels of α-linolenic acid (C18:3), while BR017 also exhibited higher protein levels (14% of total dry weight), suggesting that these may be interesting strains to be used within the biorefinery concept. Furthermore, Monoraphidium sp. BR023 showed higher lipid content (29% of total dry weight), and transesterifiable fatty acid production (43.2μgmg−1 dry weight) and lower starch levels (164μmolmg−1 dry weight) while Chlamydomonas sp. BR020 exhibited the opposite trend. The results suggest that Scenedesmus bajacalifornicus BR024, Selenastrum capricornutum BR009 and Scenedesmus obliquus BR003 represent potential strains for biomass and biodiesel production, presenting 1.81, 1.73 and 2.14mgmL−1 dry weight of biomass, respectively, and 21.6, 16.4 and 17% of total dry weight of lipid, respectively. Additionally, the two Scenedesmus species showed the highest lipid productivity (23mgL−1d−1 and 24mgL−1d−1). Overall we were able to access a new source of microalgal biodiversity and perform a robust characterization of native tropical microalgae strains leading to the identification of strains with high potential for biotechnological applications.