Fungal metabolites are known to have potent and diverse properties such as antiviral, antidiabetic, antitumour, antioxidant, free radical scavenging, and antibacterial effects which can be utilized to treat diseases. In this study, we investigated the functional activity of stereumamide A (StA) derived from a culture broth of Trichaptum fuscoviolaceum during the in vitro fertilization (IVF) of pig oocytes, to determine its effects on sperm penetration. Oocytes matured in vitro were fertilized in the absence or presence of varying concentrations of StA (0-50 μg/ml StA). When StA was directly added into the IVF medium, significantly lower fertilization rates were seen with the 20 or 50 μg/ml StA (2.0–17.5%) treatments compared with those of 10 μg/ml StA or the controls (60.9–62.3%), whereas StA had no influence on the survival of oocytes and spermatozoa throughout the IVF process. For evaluating the control of sperm entry, mature oocytes were pre-incubated in a medium containing 20 μg/ml StA for 1 h, and then IVF was subsequently performed. The incidence of polyspermy was significantly reduced when oocytes were pre-incubated with StA (15.0% vs. 50.4–57.5% in controls). In conclusion, sperm penetration was inhibited in the medium in the presence of StA during IVF, while StA did not affect sperm motility and fertility competence. Fertilization was controlled when mature oocytes were incubated with StA prior to IVF, suggesting the possible use of the fungal metabolite in assisted reproductive technology for humans and animals.