Mixotrophic protists are capable of acting both as primary producers and primary consumers at the base of the aquatic food web, thus constituting key organisms in ecosystems where they are abundant. However, their identity, abundance, ecological dynamics, and biogeochemical impact in aquatic ecosystems remain understudied in comparison to classically demarcated autotrophs or heterotrophs. In this study, we make use of fluorescently labeled prey and fluorescence-activated cell sorting to taxonomically identify actively-feeding individual mixotrophic flagellates from lake water. Replicated experiments were carried out to assess the performance of three different fluorescently labeled prey types and a fluorescent dye targeting food vacuoles. In the experiments, water from an oligotrophic lake was exposed independently to each type of reporter and cells were individually sorted based on fluorescent signals for predation and chlorophyll a. A total of 927 individual single cells were successfully recovered, with all fluorescent reporters exhibiting high sensitivity for putative mixotrophic taxa: overall, 87% of the occurrences could be assigned to dictyochophytes, 9% to chrysophytes, and 3% to dinoflagellates. As a result, we were able to detect cryptic diversity within pedinellid algae and report a Prorocentrum-like freshwater occurrence. We argue that this procedure can be a valuable tool to uncover relevant and unexpected active mixotrophic species in a wider range of aquatic environments, and could easily be coupled to other techniques to describe the finer details of the trophic status of aquatic microbial communities.