Marine primary productivity supports food webs and ecosystem health, driving large-scale animal distribution patterns in the ocean. Primary productivity is also a fundamental process in the biological pump, which sequesters inorganic carbon from the atmosphere through photosynthesis and transports it to the ocean interior where it can be stored for millennial or greater timescales. Presently, different methods for quantifying productivity disagree with each other, presenting a major research challenge. To understand how climate change may impact the biosphere, it is necessary to continuously improve methods for quantifying primary productivity. The isotopic composition of dissolved oxygen in the ocean can be used as a constraint on oxygen production and therefore, be used to quantify marine primary productivity through the carbon-oxygen stoichiometry of photosynthesis. To distinguish newly produced O2 from O2 already present in the atmosphere, the 18O/16O and 17O/16O ratios of dissolved oxygen can be used. This approach is termed the “triple-oxygen isotope (TOI) method” and it relies on laboratory studies of the behavior of oxygen isotope ratios during photosynthesis to constrain field measurements. Despite the potential of the TOI method, major knowledge gaps remain. For example, nutrient availability affects the photosynthesis activity of microorganisms and is heterogeneous across global oceans. Therefore, understanding how oxygen isotope fractionation may vary under nutrient str ess is crucial for correctly interpreting field measurements. To address this knowledge gap, this study examines the effect of nitrate availability on the isotopic composition of cyanobacterial photosynthetic oxygen. Freshwater cyanobacteria Synechocystis PCC 6803 are inoculated in media with different nitrate concentrations. The cyanobacterial photosynthetic oxygen is collected and its TOI composition is analyzed. Additionally, this study generates new data on the clumped isotope composition of photosynthetic oxygen, which is a parameter describing the degree of randomness in the distribution of rare oxygen isotopes (17O and 18O) in O2 molecules. Results suggest that there is larger oxygen isotope fractionation when cyanobacteria experience more nitrate limitation, and that the TOI and clumped isotope composition of photosynthetic oxygen can be utilized together to constrain gross oxygen production and provide information on the mechanism of cyanobacterial photosynthesis.