High-throughput sequencing technologies have revolutionized microbial diversity studies, shedding light on the oceans’ plankton evolution, distribution, and biological activity. Whereas marine prokaryotes have been more extensively studied and specific methods developed, the research on microbial eukaryotes (protists) is falling behind, with major groups still largely unknown regarding their ecology and function. Because of numerous anthropogenic pressures, it is increasingly important to highlight the functional roles of protists in marine ecosystems. This review outlines the practices, challenges, and opportunities of high-throughput sequencing approaches (i.e., metabarcoding, metagenomics, and metatranscriptomics) to disentangle evolutionary, ecological, and functional aspects of protists in the ocean. These multidimensional approaches allow us to move from the classic picture of microbial biogeography towards functional microbial biogeography, explicitly highlighting the role of protists therein. We provide resources for functional classification and reflect on the current and future potential. We outline aspects of detecting and describing ecosystem changes at the species, population, and community levels, advancing methodological approaches for studying taxonomic diversity towards functional and evolutionary biodiversity concepts, seeking a more complete understanding and monitoring of ocean ecosystems.