Bovine rotaviruses (BRVs) are significant causative agents of severe diarrhea in newborn calves, resulting in substantial economic losses in the livestock industry. Inhibition of bovine rotavirus using extracts prepared from a Cucurbitaceae plant, which contains trypsin protease inhibitors, might offer a potential anti-rotaviral effect in vitro. Ecballium elaterium (E. elaterium) belongs to the Cucurbitaceae family, indigenous to the Mediterranean, contains E. elaterium trypsin isoinhibitors (EETIso), and has been used in traditional medicine. This study aimed to evaluate the in vitro efficacy of E. elaterium extract against bovine rotavirus infections. Ethanol extracts were prepared from E. elaterium seeds and fruit juice, and their non-toxic concentrations were determined using MA-104 cells. The cells were infected with bovine rotavirus in the presence of E. elaterium extract. The results demonstrated a significant decrease in the rotavirus titer in vitro upon treatment with the E. elaterium extract, suggesting its potential as a therapeutic agent against bovine rotavirus-induced diarrhea in calves. The utilization of E. elaterium extract may contribute to reduced calf mortality, lower medication costs, and improved economic value in cattle farming.