Nitrate contamination of groundwater poses serious risks to drinking water safety, particularly in agriculturally impacted regions, while conventional treatment technologies remain costly and energy intensive. This study evaluates microalgae-based nitrate removal from groundwater, with particular emphasis on the role of precultivation strategy in controlling nitrate removal kinetics, biomass productivity, and CO₂ fixation. Chlorella vulgaris and Scenedesmus sp. were precultured in Blue-Green 11 and Bold’s Basal Medium and subsequently cultivated in nitrate-contaminated groundwater (50 mg NO₃⁻–N/L) using photobioreactors. Both species effectively removed nitrate; However, treatment performance was strongly dependent on species and precultivation medium. BG-11–precultured Scenedesmus sp. demonstrated the highest and fastest nitrate removal, reducing NO₃⁻–N below the drinking water standard (10 mg/L) within 78 h and achieving 99% removal after 96 h. BG-11–precultured Chlorella vulgaris reached 83.4% removal over the same period. BBM-precultured Scenedesmus sp. also achieved high nitrate removal (96%), but with slower kinetics, while BBM-precultured Chlorella vulgaris showed the lowest removal efficiency (87.8%). Enhanced nitrate removal was directly associated with increased biomass accumulation and CO₂ fixation, with BG-11–precultured Scenedesmus sp. attaining the highest biomass (0.45 g/L) and CO₂ fixation rate (20.68 g/L/d). This study demonstrates that precultivation strategy is a critical control parameter for microalgal groundwater remediation and identifies BG-11–conditioned Scenedesmus sp. as a highly effective candidate for integrated nitrate removal and carbon sequestration in real groundwater systems.