The composition of brackish groundwater from Brazilian backlands contains important elements necessary for metabolism in microalgae. This study evaluated the use of 100% brackish groundwater with different amounts of Zarrouk nutrients for Spirulina sp. LEB 18 cultivation. The growth parameters and biomass composition, including the concentrations of proteins, carbohydrates, ash, lipids, and fatty acids, were evaluated. The best growth parameter results were obtained in the assay using 100% brackish groundwater and only 25% of Zarrouk nutrients, which were equal to those obtained for the control culture. The concentrations of carbohydrates and polyunsaturated fatty acids were increased by as much as 4- and 3.3-fold, respectively, when brackish groundwater was used in the cultures. The lipid profile demonstrated that the biomass had the potential for use in biodiesel production. The use of brackish groundwater is a sustainable, economical way to obtain high-quality biomass for different applications during Spirulina sp. LEB 18 cultivation.