Microalgae have the ability to assimilate atmospheric carbon dioxide and organic and inorganic pollutants while simultaneously producing a range of useful intracellular metabolites. Harvesting and concentrating microalgal suspensions, which is commonly performed via coagulation, flocculation, or filtration, is complex, owing to the dynamic character of the cells. The availability of nitrogen to the cells plays a crucial role in the synthesis of intracellular products and in the release of extracellular organic matter. These are expected to have an extensive impact on the harvesting/dewatering behavior. In this study, the role of nitrogen availability in the production and release of extracellular matter was investigated by characterizing the extracellular organic matter composition and association with the cells for three commercially relevant microalgae: Nannochloropsis salina, a marine Chlorella sp., and Haematococcus pluvialis. The results showed that nitrogen limitation reduced the amount of extracellular matter on a per cell basis, despite the bulk concentration increasing due to the increase in the total biomass surface area. In particular, the protein content of the extracellular material was found to decrease across all three species upon nitrogen deprivation. However, there was significant variation in the amount and composition of the extracellular organic matter between the microalgae. This study provides new knowledge of the variations of extracellular substances in relation to microalgae species and cultivation conditions. This has important implications for species selection and optimization of microalgae production, harvesting, and dewatering processes.