Natural products of plant origin were investigated for their biological, nutritional, and pharmacological properties. Previous studies on the bioactivity of polyphenols extracted from maize tissues (Zea mays, L.) demonstrated tumor growth inhibition and immune function stimulation, as well as cell proliferation inhibition, and apoptosis induction. The present in vitro study evaluated the antitumor effect of the polyphenolic extract (PE) of Z. mays leaves (Rajado 8 Carreiras creole variety) on a neoplastic cell line of murine melanoma (B16F10). Assays related to the determination of the PE chemical profile, as well as its impact on biological mechanisms, were conducted. High performance liquid chromatography analysis of PE revealed the presence of chlorogenic acid as the major compound (24.21 ± 1.2 ug/mL). Further in vitro assays demonstrated that PE reduced cell viability up to 77 ± 0.1% (7 ug/mL, 24 h) with an IC50 of 3.5 ug/mL. Additionally, PE induced apoptotic cell death by up to 29 ± 1.7% (5 ug/mL, 24 h), although the cell proliferation rate did not differ statistically from the control group. This finding suggests that the tested concentrations may be toxic, leading to the induction of cell apoptosis. Concerning its ability to form clones, the B16F10 line exhibited sensitivity to PE, reducing colony formation by up to 99 ± 0.5% (5 ug/mL, 24 h). PE also induced changes in the pattern of actin and vinculin structures and in the distribution of focal adhesion points, correlating with the increase of the administered doses. The data set suggests that PE from Z. mays promotes alterations in the structure of the cytoskeleton, resulting in direct effects on the ability of cells to adhere and, consequently, the growth of cell colonies, i.e., clones, leading to the induction of apoptosis.