Applications of solutions with various organic acids have been widely demonstrated as effective disinfectants on lettuce. However, agronomic techniques of improving the concentration of internal organic acids in lettuce are not well investigated. Hereon, changes in growth, antioxidants, and organic acids of baby lettuce (Lactuca sativa L. var. longifolia) resulting from different levels of nitrogen fertilizer (0.10, 0.14, 0.18, or 0.22 g/5 kg soil) and water supply (300, 600, or 900 mL) were investigated. The pot experiment was conducted under a net house at North-West University (Mafikeng Campus), South Africa. Data on growth parameters (number of leaves and leaf area) and chlorophyll concentration were sampled weekly until leaf organic acids (citric, malic, and tartaric), total antioxidant compounds (TAO), as well as dry matter content, were measured at harvest. Reducing the amount of water supplied to baby lettuce significantly (p < 0.05) increased the chlorophyll concentration, leaf area, TAO, citric acid, and malic acid. The number of leaves and the leaf tartaric acid concentrations were increased by increasing the treatment levels. It was conclusive that decreasing water supply increases the organic acids regardless of the nitrogen level.