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Aquatic ecologists are integrating mixotrophic plankton – here defined as microorganisms with photosynthetic and phagotrophic capacity – into their understanding of marine food webs and biogeochemical cycles. Understanding mixotroph temporal and spatial distributions, as well as the environmental conditions under which they flourish, is imperative to understanding their impact on trophic transfer and biogeochemical cycling. Mixotrophs are hypothesized to outcompete strict photoautotrophs and heterotrophs when either light or nutrients are limiting, but testing this hypothesis has been hindered by the challenge of identifying and quantifying mixotrophs in the field. Using field observations from a multi-decadal northern North Atlantic dataset, we calculated the proportion of organisms that are considered mixotrophs within individual microplankton samples. We also calculated a “trophic index” that represents the relative proportions of photoautotrophs (phytoplankton), mixotrophs, and heterotrophs (microzooplankton) in each sample. We found that the proportion of mixotrophs was positively correlated with temperature, and negatively with either light or inorganic nutrient concentration. This proportion was highest during summertime thermal stratification and nutrient limitation, and lowest during the North Atlantic spring bloom period. Between 1958 and 2015, changes in the proportion of mixotrophs coincided with changes in the Atlantic Multi-decadal Oscillation (AMO), was highest when the AMO was positive, and showed a significant uninterrupted increase in offshore regions from 1992-2015. This study provides an empirical foundation for future experimental, time series, and modeling studies of aquatic mixotrophs.