Auto-inhibition is widely observed in the microalgal media reuse, but auto-inhibitors in the reused culture media are not well understood. In the current study, microalgal media reuse experiments, target-directed organic matter separation and identification via gas chromatography-mass spectrometry (GC-MS) were conducted to elucidate the growth inhibitors in the reused media of the Chlorophyta Scenedesmus acuminatus. Results indicated growth inhibitors in the reused media belonged to small molecule dissolved organic matter that was mainly produced at the declining growth phase. Using an ultraviolet detector-guided resin separation procedure, dissolved organic matter in the reused media was partitioned into 13 fractions. Growth inhibition tests showed that inhibitors were in the fraction 8. GC-MS analysis revealed that fraction 8 contained fatty acid, phenol, alicyclic, and thioester compounds along with hexadecanoic acid and octadecanoic acid as its major components. Further experiments verified the existence of hexadecanoic acid and octadecanoic acid in the original reused media, and commercial standards also confirmed the growth inhibition to S. acuminatus by hexadecanoic acid and octadecanoic acid. This is the first study to confirm that microalgae can produce hexadecanoic acid and octadecanoic acid which inhibit their own growth. This study establishes an innovative target-directed method to fractionate, screen and identify compounds in dissolved organic matter. Meanwhile, the results of this study could be conducive to develop effective ways to reuse microalgal culture media and save water in microalgal cultivation.