Microalgae have been identified as one of the most promising sources of novel bioactive compounds for biomedical applications, the food industry, and cosmetics. In the last decade, several biotechnological developments have facilitated the identification of a growing number of compounds as well as the study of optimal microalgae culture conditions for the production of biomass enriched in specific molecules of interest. In this study, two common commercial marine microalgae (Nannochloropsis oculata and Porphyridium purpureum) were cultured in standard and nutrient-stressed conditions and the obtained biomass extracts were assessed for their potential to inhibit cancer cell proliferation and migration as well as their antioxidant activity. Results from viability in 2D and 3D cancer cell models showed an enhancement of the antitumour activity of P. purpureum in the 3D model compared to 2D, together with a greater capacity to reduce the migration capacity of cancer cells with the biomass from nutrient-stressed conditions, whereas the antioxidant activity of N. oculata decreased when exposed to nutrient-stressed conditions. To date, this is one of the few studies that proves that controlled changes in large-scale culturing conditions such as nutrient depletion have a relevant impact in the bioactivity of the biomass on cancer cells.