The primary source of biodiesel obtainment in Brazil is concentrated in oleaginous vegetables, especially soy. However, there is a great hold-up caused by competition with the food industry, which makes the search for alternative pathways that do not compete with other industries needed. The goal of this study was to investigate the biotechnological potential of cyanobacteria, which were grown in two different culture media, as an alternative approach for obtaining biodiesel. For this purpose, Microcystis aeruginosa CACIAM03, CACIAM08, Synechocystis sp. CACIAM05, Lyngbya sp. CACIAM07, Cyanobium sp. CACIAM14, Leptolyngbya sp. CACIAM18, Limnothrix redekei CACIAM25 and Planktothrix pseudoagardhii CACIAM27 were evaluated. Fatty acid composition was determined using gas chromatography. Fatty acids found in all strains were identified as palmitic acid (C16:0) (7.43–38.37% content), stearic acid (C18:0) (1.44–13.82%), caproic acid (C6:0) (0.82–78.84%) and oleic acid (C18:1) (1.13–46.76%). The biodiesel quality parameters were calculated based on the fatty acid profile. Strains that showed the best values and provided better biodiesel quality were Synechocystis sp. CACIAM05 and Microcystis aeruginosa CACIAM03 that were grown in BG-11 medium. This study showed us a promising source of biodiesel production from cyanobacterial lipids based on empirical calculations of the parameters of biodiesel quality that met the fuel standards. Furthermore, our results suggest the production of fatty acids through a metabolic route, due to the change in the profile of fatty acids in both culture media, providing valuable information for future engineering to increase the percentage of fatty acids that give better biodiesel quality.