Phytoplankton-derived dissolved organic matter (DOM) sustains complex marine microbial communities, yet the role of marine fungi-particularly yeasts-remains understudied regarding their substrate preferences, enzymatic strategies, and ecological relevance. We developed a novel protocol to investigate substrate-specific growth of marine fungal isolates under defined conditions and high temporal resolution. Using the β-1,3-glucan laminarin-a major marine storage polysaccharide of phytoplankton-and its oligomeric and monomeric breakdown products, we characterized growth and substrate utilization profiles of eleven marine yeast isolates from the epipelagic zone at Helgoland Roads, North Sea. Statistical analyses of growth kinetics distinguished four ecotypes with distinct substrate utilization patterns, quantified via phenol-sulfuric acid assays. Fluorophore-assisted carbohydrate electrophoresis (FACE) revealed the lack of endo-laminarinase activity, suggesting laminarin degradation depends on exo-acting enzymes. FACE also revealed a high diversity of short-chained laminarin-based intermediates accumulating over time, demonstrating that yeasts enhance chemical complexity during laminarin degradation and may fuel other microbes within the microbial loop. Representatives of each yeast ecotype were found to match abundant operational taxonomic units (OTU) in sequence similarity analyses of epipelagic mycoplankton datasets. This supports their ecological success and diverse substrate strategies. Rather than acting solely as opportunists, these yeasts may actively shape DOM turnover and carbon cycling within the microbial loop. Our study highlights a robust experimental approach for resolving functional diversity among marine yeasts and underpins their potential role in maintaining chemical diversity and substrate cross-feeding in the microbial loop.