In 2006, species of Cochlodinium geminatum were first recorded as new, harmful algal bloom (HAB)-forming dinoflagellate, in the Pearl River Estuary, China. Previous studies indicated that allelopathy (the release of chemical compounds) is an important competitive strategy for C. geminatum in inhibiting the growth of phytoplankton competitors, however, little is known about the allelochemicals produced by this species. In this study, allelopathic compounds were characterized using a series of chromatographic techniques. Methanol extracts from cultures showed significant inhibitory effects towards the co-occurring phytoplankton, Akashiwo sanguinea, and potent hemolytic activity against rabbit erythrocytes. Solid phase extraction (SPE) fractions from a reverse phase SPE column, exhibited inhibitory and synergistic effects on A. sanguinea growth, of which fractions eluted in 80% and 100% methanol showed the strongest allelopathic activity. These fractions were further purified using reverse phase high performance liquid chromatography (HPLC), and six fractions were collected. Three fractions displayed strong inhibitory effects towards A. sanguinea, with 14%–25% inhibitory rates, but a higher 35% inhibitory rate was observed when fractions were combined, suggesting allelochemicals had synergistic allelopathic effects. Allelopathic activity also exhibited time dependent reduction when stored at -20°C. Furthermore, trypsin digestion and dialysis assays indicated C. geminatum produced multiple allelopathic compounds. Our study demonstrated that species of C. geminatum produced and released multiple allelopathic compounds of low polarity and sufficient hydrophilicity, which elicit important competitive advantages by inhibiting co-occurring phytoplankton, and promoting population growth.