The present investigation was carried out to evaluate the antifungal activities of Phyllanthus polyphyllus L. leaf extract, to isolate its active constituent 4-o-methyl gallic acid (4-o-MGA), and to determine the antioxidant and antiaflatoxigenic properties. The bioassay-guided fractionation of methanol extract led to the isolation of active compound 4-o-methylgallic acid. The methanol extract showed the highest amount of phenolic content (290 mg GAE/g dry extract), which indicated the involvement of phenolic compounds in the radical scavenging activity observed by the methanol extract. The antioxidant capacity of 4-o-MGA was greater than the standard butylated hydroxytoluene, followed by methanol extract. The concentration-dependent growth inhibitory activity was observed against the tested fungi, in which the field fungi were susceptible, while the storage fungi were found to be more resistant including aflatoxigenic A. flavus. A correlation was observed between fungal biomass and aflatoxin production in control and treatment, there was a decrease in biomass of A. flavus and aflatoxin production with increasing concentration. The aflatoxin production was completely inhibited in vitro by methanol extract at 1 mg/mL and 4-o-MGA at 2 mg/mL, but the mycelial growth was not inhibited completely. The inhibition of aflatoxin production was relatively higher than the mycelial growth inhibition of A. flavus, such behaviors might have been determined by the presence of hydrolysable tannin 4-o-MGA in the extract, which is known to inhibit the aflatoxin biosynthesis. The significant antioxidant and aflatoxin inhibitory activities of P. polyphyllus could be exploited for its application in preventing oxidative deterioration and fungal spoilage of food products.