Protozoan grazing is central to marine microbial food webs, but its role in organic matter cycling is commonly framed mainly as bacterial mortality and trophic transfer. Grazing, excretion, and incomplete digestion also regenerate dissolved organic matter (DOM), potentially changing both bacterial substrates and the communities responsible for subsequent DOM transformation. How these top-down and bottom-up effects interact remains poorly resolved. We integrated three complementary experimental systems: resource-depletion macrocosms to examine the environmental dependence of prey selection; standardized ciliate–bacteria cultures coupled with fluorescence spectroscopy and ultrahigh-resolution molecular characterization to assess grazer-specific DOM formation; and utilization experiments with a bacterial isolate and natural microbial assemblages to evaluate community-dependent DOM reworking. Resource depletion altered the responses of high- and low-nucleic-acid prokaryotes and patterns of selective grazing, showing that top-down regulation depends on prey physiological state and environmental context. In standardized cultures, both Euplotes and Uronema reduced heat-killed bacterial prey by one to two orders of magnitude while increasing in abundance. The humic-like fluorescent component C2 followed the pattern prey-only control < Euplotes < Uronema. Molecular analysis showed preferential loss of highly unsaturated formulas, decreased overall unsaturation, and relative enrichment of CHON and protein- or carbohydrate-like formulas, whereas aromaticity and nominal carbon oxidation state changed little. Thus, DOM formation during grazing involved grazer-specific optical signatures and selective molecular redistribution rather than uniform oxidation or simple bulk accumulation. The subsequent utilization of grazing-associated DOM depended on the recipient community. Natural microbial assemblages showed concentration-dependent growth, with high-nucleic-acid bacteria responding rapidly, whereas the original prey isolate showed little short-term net-growth response. DOM bioavailability therefore emerged from interactions between substrate composition and community metabolic capabilities. Together, these findings show that protozoa couple selective prey removal with grazer-dependent DOM regeneration and community-dependent reworking. This framework extends their role beyond bacterial mortality and provides a process-based basis for incorporating protozoa into marine DOM cycling and the microbial carbon pump.