Microalgae are a novel source of sustainable natural antioxidants with various applications, including food preservation. To optimize antioxidant production in microalgae, we investigated the influence of nutrient limitation on antioxidant content in three species, Phaeodactylum tricornutum, Tetraselmis suecica and Chlorella vulgaris. Microalgae were cultivated in batch culture under nutrient replete, P- and N-limited conditions. Total antioxidant activity of the biomass was measured using Trolox equivalent antioxidant capacity and square wave voltammetry. Additionally, contents of carotenoids, phenolics, tocopherols and ascorbic acid were measured. Nutrient limitation, particularly N-limitation, resulted in low antioxidant content. Both phenolic and carotenoid contents were significantly reduced in nutrient-limited cultures. In contrast tocopherols and ascorbic acid levels were higher in nutrient-limited cultures, particularly under P-limitation. Our results indicate that nutrient stress is not an effective strategy to enhance overall antioxidant content in microalgae, although it may be useful to enhance production of some vitamin antioxidants such as tocopherols or ascorbic acid.