Abstract: Aspergillus flavus (A. flavus) is a well-known diffused fungus that contaminates a great number of crops used for human and animal consumption. How to effectively prevent the contamination becomes a significant problem. Camellia (Camellia oleifera Abel.) is one of the four dominant, woody, oil species in the world. Oil from camellia seeds is rich in essential fatty acids and vitamins. This oil can alleviate malnutrition by balancing human nutrition to benefit human health, and it conforms to the consumption trends of modern edible oils, along with the utilization of its by-products is getting more and more attention. In recent years, the prevention and treatment of A. flavus and AFs by plant sources and active microbial substances have attracted more attention. Our previous study shows that camellia seed cake is not susceptible to A. flavus. Based on the result, we propose that there may be a certain natural antagonistic substances that can inhibit the growth of A. flavus and the production of AFs in camellia seeds. This study was designed to evaluate the antifungal effect of the active substance in camellia seed cake on the growth of A. flavus and toxigenicity through in-vitro and in-vivo antifungal tests, to make a determination of antimicrobial substances contained in the camellia seed cake. Camellia seed cake was extracted by eighty percent methanol (v/v), the filtrate was extracted with ethyl acetate and saturated n-butanol. Among the extracts, the n-butanol phase exhibited apparent inhibition activity on the growth and aflatoxin production of A. flavus. The concentration of 100 mg/mL worked best, because the inhibitory zone diameter was 22.00 mm, the mycelial dry weight was 42.88% less than the control, both of the two indicators were significantly better than other treatments (P<0.01), and no AFs were detected. Whereas the aqueous phase and ethyl acetate phase exhibited weak antifungal activity and no activity, respectively. In addition, the n-butanol phase inhibited the production of aflatoxin B1 (AFB1) of A. flavus effectively, AFB1 was not detected in all concentration treatments. The main chemical component of n-butanol extract phase of eighty percent methanol extract in camellia seed cake was tea saponin, the research showed that tea saponin had no inhibitory effect on A. flavus. Therefore, it was speculated that the other active component (s) in the n-butanol extract phase had inhibitory effect on A. flavus. n-Butanol extract phase was separated and purified by means of AB-8 macroporous absorbent column chromatography, among the macroporous resin fraction of 0%, 25%, 50%, 75%, and 100% ethanol (v/v), the 75% ethanol fraction showed the highest antifungal activity, the concentration of 100 mg/mL worked best, because the inhibitory zone diameter was 163.7 mm, the mycelial dry weight was significantly lower than the control, both of the two indices were significantly superior to other treatments (P<0.01), and AFs were undetected. The 75% ethanol fraction was assayed by ultra-performance liquid chromatography- quadrupole time-of-fight mass spectrometry (UPLC-Triple-TOF/MS). The three compounds were identified which were 1) kaempferol-3-O-β-D-glucopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-galactopyranoside; 2) kaemferol-3-O- 2-O-β-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl-β-D-glucopyranoside; and 3) kaempferol-3-O-6-trans-p-coumaroyl)- β-D-glucopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-galactopyranoside. This study provides a reference and a guide for research and development of an antifungal agent for A. flavus, and meanwhile broadens utilization of the by-products of camellia seed.