Occurrence of cyanobacterial harmful algal blooms (CyanoHAB) can induce considerable patchiness in the concentration and bioavailability of dissolved organic matter (DOM), which could influence biogeochemical processes and fuel microbial metabolism. In the present study, a laboratory 4-stage plug-flow bioreactor was used to successfully separate the CyanoHAB-derived DOM isolated from the eutrophic Lake Taihu (China) into continuum classes of bioavailable compounds. A combination of new state-of-the-art tools borrowed from analytical chemistry and microbial ecology were used to characterize quantitatively the temporary evolution of DOM and to get deeper insights into its bioavailability. The results showed a total 79% dissolved organic carbon loss over time accompanied by depletion of protein-like fluorescent components, especially the relatively hydrophilic ones. However, hydrophilic humic-like fluorescent components exhibited bioresistant behavior. Consistently, ultrahigh resolution mass spectrometry (FTICR-MS) revealed that smaller, less aromatic, more oxygenated, and nitrogen-rich molecules were preferentially consumed by microorganisms with the production of lipid-like species, whereas recalcitrant molecules were primarily composed of carboxylic-rich alicyclic compounds. Moreover, the bioavailability of DOM was negatively correlated with microbial community diversity in the bioreactor. Results from this study provide deeper insights into the fate of DOM and relevant biogeochemical processes in eutrophic lakes.