Many marine microbial eukaryotes combine photosynthetic with phagotrophic nutrition, but incomplete understanding of such mixotrophic protists, their functional diversity and underlying physiological mechanisms limits assessment and modeling of their roles in present and future ocean ecosystems. We developed an experimental system to study responses of mixotrophic protists to availability of living prey and light, and used it to characterize contrasting physiological strategies in two stramenopiles in the genus Ochromonas. We show that oceanic isolate CCMP1393 is an obligate mixotroph, requiring both light and prey as complementary resources. Interdependence of photosynthesis and heterotrophy in CCMP1393 comprises a significant role of mitochondrial respiration in photosynthetic electron transport. In contrast, coastal isolate CCMP2951 is a facultative mixotroph which can substitute photosynthesis by phagotrophy and hence grow purely heterotrophically in darkness. In contrast to CCMP1393, CCMP2951 also exhibits a marked photoprotection response that integrates non-photochemical quenching and mitochondrial respiration as electron sink for photosynthetically-produced reducing equivalents. Facultative mixotrophs similar to CCMP2951 might be well adapted to variable environments, while obligate mixotrophs similar to CCMP1393 appear capable of exceptionally resource efficient growth in oligotrophic ocean environments. Thus, the responses of these phylogenetically close protists to the availability of different resources reveals niche differentiation that influences impacts in food webs and leads to opposing carbon-cycle roles.