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Abstract Freshwater ecosystems are increasingly at risk of experiencing toxin-producing cyanobacterial blooms during the winter due to anthropogenic nutrient loading and climate change. However, understanding how increased light, temperature and nutrient levels impact cyanotoxin production during the winter is limited, as most research has historically focused on blooms during the summer and fall. We conducted 2 × 2 × 2 incubation experiments in February and March to test the individual and interactive effects of light intensity (50 and 150 μmol m−2 s−1 PAR), elevated temperature (+3°C), and nitrogen and phosphorus enrichment on microcystin concentrations in a Planktothrix agardhii-dominated community sampled from Grand Lake Saint Mary’s, a hypereutrophic Ohio reservoir. Microcystin concentration significantly increased with elevated temperature in both months. In February, low light also promoted higher microcystin concentrations, particularly when combined with elevated temperature and nutrient enrichment. In March, nutrient enrichment had individual and interactive effects with temperature that caused higher microcystin concentrations. These results demonstrate that toxin-producing cyanobacteria are active in winter and that climate-driven changes in environmental conditions can interactively increase total toxin concentrations in the water column, even in the non-growing season.