Objectives: This study analyzed the antiproliferative potential of Eugenia uniflora L. leaf extracts against cervical cancer and non-cancerous cell lines. Methods: The extracts were prepared by maceration using hexane (EUH), dichloromethane (EUD), and ethyl acetate (EUA). Their cytotoxic potential was evaluated through MTT assays, wound healing assays, and flow cytometry. To identify classes of secondary metabolites, total phenolic and flavonoid contents were quantified using spectrophotometric methods, and individual metabolites were tentatively identified by LC-MS/MS. Results: EUH, EUD. and EUA exhibited cytotoxicity in HeLa cells, with IC₅₀ values of 63.03 μg/mL, 33.79 μg/mL, and 38.38 μg/mL, respectively. Due to their lower IC₅₀ values, the EUD and EUA fractions were selected for further investigation. EUA and EUD inhibited cell migration at all the time points tested and altered the cell cycle. Twenty-eight compounds were tentatively identified in E. uniflora L. leaf extracts based on the interpretation of their fragmentation patterns and molecular formulas obtained from mass spectrometry. Conclusions: The EUD and EUA extracts appear to modulate the metabolism of cervical cancer cells, leading to cell cycle arrest and inhibition of cell migration. Flavonoids and other phenolic compounds are likely responsible for these observed biological effects.