Functional substances from mackerel wastewater were biodegraded and tested for inhibitory activity against harmful algal blooms (HABs) that are detrimental to aquaculture. The supernatant from a 48 h culture of mackerel wastewater had a half-maximal inhibitory concentration of 0.54?0.68 mg/ml for the three tested HAB organisms (Heterocapsa triquetra, Alexandrium fundyense and Prorocentrum minimum). This inhibitory effect was not observed form 48-h biodegraded nutrient broth culture supernatant, indicating inhibitory substances did not originate from bacterial metabolites. Rough estimation of molecular weight using ultrafiltration indicated that the inhibitory substance was less than 2 kDa, and it did not inhibit the non-HAB organism (Skeletonema costatum), which is essential for preserving marine ecosystems in real-world applications. Furthermore, this inhibitory effect against HABs was not observed for biodegraded culture supernatants of other protein sources (skim milk and okara), indicating that the inhibitory substances were derived from a particular source present only in raw mackerel wastewater. To the best of our knowledge, this is the first report describing the selective inhibitory effects of the culture supernatant of mackerel wastewater against HABs.