Existing research shows that the presence of algogenic organic matter (AOM) in recycled media can inhibit the growth of algae. However, the characteristics and occurrence of the inhibitors are not well understood. In this study, changes in the algogenic organic matter present in culture media and the influence of these changes on the recultivation of Scenedesmus acuminatus were investigated. A hydrophobic humic substance was then extracted from the recycled medium, and its inhibitory effects on the growth of Scenedesmus acuminatus were assessed. The efficiency of the removal of the humic substance from the recycled media using granular activated carbon (GAC) treatment was evaluated. The results showed that the later the growth phase at which the recycled media was harvested, the stronger the growth inhibition was. In addition, it was found that the percentage of total carbohydrates, fatty acids, and proteins in the algogenic organic matter decreased with the prolongation of growth phase, while the percentage of the hydrophobic humic substance in algogenic organic matter increased as the algae gradually went into the declining phase. Furthermore, an inhibitory effect of the hydrophobic humic substances on the growth of Scenedesmus acuminatus was identified. The effectiveness of inhibitor removal using granular activated carbon column adsorption was confirmed. The results of this study can be applied to the sustainable utilization of water and nutrients in the mass production of microalgal biomass.