Viruses represent the primary etiologic agents (70-80%) of acute diarrheal disease (ADD), and rotavirus (RV) is the most relevant one. Currently, four rotavirus vaccines are available. However, these vaccines do not protect against emerging viral strains or are not available in low-income countries. To date, there are no approved drugs available against rotavirus infection. In this study, we evaluated the in vitro anti-rotaviral activity and intestinal toxicity of a phytotherapeutic prototype obtained from Achyrocline bogotensis (Kunth) DC. (PPAb); medicinal plant that contains compounds that inhibit the rotavirus replication cycle. Virucidal and viral yield reduction effects exerted by the PPAb were evaluated by immunocytochemistry and flow cytometry. Furthermore, the toxic impact of the PPAb was evaluated in polarized human intestinal epithelial C2BBe1 cells in terms of cytotoxicity, loss of cytoplasmic membrane asymmetry, and DNA fragmentation by MTT and fluorometry. PPAb concentrations under 0.49 mg/mL exerted significant virucidal and viral yield reduction activities, and concentrations under 16 mg/mL neither reduced cell viability, produced DNA fragmentation, nor compromised the C2BBe1cell membrane stability after 24-h incubation. Based on these results, the evaluated phytotherapeutic prototype of Achyrocline bogotensis might be considered as a promising alternative to treat ADD caused by rotavirus.