Protists are traditionally described as either phototrophic or heterotrophic, but studies have indicated that mixotrophic species, organisms that combine both strategies, can have significant impacts on prey populations in marine microbial food webs. While estimates of active mixotroph abundances in environmental samples are determined microscopically by fluorescent particle ingestion, species identification is difficult. We developed SYBR-based qPCR strategies for three Antarctic algal species that we identified as mixotrophic. This method and traditional ingestion experiments were applied to determine the total mixotroph abundance in Antarctic water samples, to ascertain the abundance of known mixotrophic species, and to identify environmental variables that impact the distribution and abundance of these species. Despite differences in sampling locations and years, mixotroph distribution was strongly influenced by season. Environmental variables that best explained variation in the individual mixotroph species abundances included temperature, oxygen, date, fluorescence, conductivity, and latitude. Phosphate was identified as an additional explanatory variable when nutrients were included in the analysis. Utilizing culture-based grazing rates and qPCR abundances, the estimated summed impact on bacterial populations by the three mixotrophs was usually < 2% of the overall mixotrophic grazing, but in one sample, Pyramimonas was estimated to contribute up to 80% of mixotrophic grazing.