Marine dissolved organic matter (DOM) is a key component of the global carbon cycle. While marine DOM properties are usually characterized by chemical approaches, an effort to integrate its variations in an ecological perspective is needed. Heterotrophic prokaryotes being the main consumers of marine DOM, targeting their responses to varying DOM properties provides an effect-based characterization of DOM properties. Dilution experiments of natural marine prokaryotic assemblages were used to assess the spatio-temporal heterogeneity of DOM properties in the coastal environment through its potential to stimulate heterotrophic prokaryotic growth. Reduced top-down pressures allowed to repeatedly evidence a stronger bottom-up stimulation potential of heterotrophic prokaryotes’ net growth with DOM collected in harbours under strong human and continental influence compared to DOM collected in more open coastal sites. Shedding the light on high spatial heterogeneity of DOM properties that corresponded only partly to dissolved organic carbon concentrations, this experimental approach represents a simple and reproducible method to improve our understanding of the marine DOM cycle.