Background: This study aimed to evaluate the antiproliferative effect of Cnicus benedictus floral extract on cervical cancer cells under in vitro conditions. Additionally, the study investigated the potential synergistic or antagonistic interactions between the plant extract and Vinblastine. Methods: The HeLa cell line was used to assess the cytotoxic effects of Cnicus benedictus floral extract, vinblastine and their combination. The tested concentrations ranged from 1 to 10,000 µg/mL, evaluated at 24 and 72 hours of incubation. Drug interaction analysis was performed using the Compusyn software to calculate the Combination Index (CI) and Dose Reduction Index (DRI). Results: The findings demonstrated that the plant extract significantly inhibited cervical cancer cell proliferation in a concentration- and time-dependent manner. A similar trend was observed for the combination treatment. In contrast, the cytotoxic effect of vinblastine was primarily time-dependent. Drug interaction analysis revealed variable effects depending on concentration. At lower concentrations, the combination exhibited synergistic effects, particularly at 24 hours of incubation. However, moderate and higher concentrations showed antagonistic interactions. The Dose Reduction Index indicated a favorable reduction in vinblastine concentration at 24 hours when used in combination with the plant extract. Conversely, at 72 hours, both vinblastine and most extract concentrations demonstrated an unfavorable dose reduction profile. Conclusion: The alcoholic extract of Cnicus benedictus exhibited significant antiproliferative activity against cervical cancer cells, comparable to vinblastine. The combination treatment showed dual interaction patterns, with synergism at lower concentrations and antagonism at moderate to higher concentrations. Furthermore, the results suggest that the plant extract may exert both cell cycle–specific and non-specific cytotoxic effects, whereas vinblastine predominantly acts through cell cycle–specific mechanisms.