Allelopathy by hydrophytes can be utilized to control algal blooms. This study was conducted to investigate the allelopathic effect (inhibition) of Scenedesmus quadricauda on Microcystis flos-aquae. When M. flos-aquae was co-cultured with S. quadricauda, the secretion of high-MW biopolymer by M. flos-aquae was inhibited by S. quadricauda. We further identified the allelochemicals and found that 4-tert-butylpyrocatechol (TBC) was the main active ingredient that could inhibit the growth of M. flos-aquae. When the dose of TBC was larger than 0.2 mg/L, almost all of the M. flos-aquae died. Additionally, TBC was found to suppress the growth of M. flos-aquae by disturbing the synthesis and secretion of proteins and polysaccharides and harming the chlorophyll to affect the light harvesting of algal cells. Therefore, TBC has the potential for use as a potential and promising algaecide to restrain the biomass of M. flos-aquae.