1H−2,3,3a,8b-tetrahydrocyclopenta[b]benzofuran, also named Rocaglamide (RocA), has gained recognition for its potential as a plant-derived therapeutic agent in cancer treatment. This study investigates its effects on breast cancer cells, specifically MDA-MB-231 human breast adenocarcinoma cells. Rocaglamide's impact was comprehensively evaluated through a series of experimental procedures. Cell viability was assessed using the MTT assay, which revealed a dose- and time-dependent reduction in cell viability, with all concentrations reducing viability to approximately 50% after 24 hours (p < 0.001), decreasing further to ~25% after 48 hours (p < 0.001), and ultimately reaching ~20% or lower by 72 hours across all concentrations (p < 0.001). Following this, apoptosis induction was examined through Annexin V-FITC staining and flow cytometric analysis, showing an increase in late apoptosis from 0.6% in controls to 2.9% in treated cells (p < 0.05) and necrosis from 7.2% to 11.7% (p < 0.005), resulting in a total cell death rate of ~15% in treated cells compared to 7.9% in controls. Cell migration was assessed by using the wound-healing assay, revealing significant inhibition of wound closure, with treated cells showing only 3.2% closure compared to 12% in controls at 24 hours, and 7.3% compared to 17.4% at 48 hours (p < 0.005). Additionally, morphological examination showed progressive changes, with untreated cells displaying typical spindle-like shapes, while RocA-treated cells exhibited rounding, shrinkage, and severe fragmentation at higher concentrations (150–500 nM). Cell cycle analysis by Propidium Iodide (PI) staining indicated alterations in phase distribution, with the proportion of cells in G1 decreasing from 55% in controls to 46% in treated cells, and the combined S and M phase populations increasing from 38% to 44%, suggesting impaired proliferation. Furthermore, protein expression levels assessed using the Human XL Oncology Array demonstrated downregulation of VEGFA (−39%), AXL (−13%), and PAI-1 (−34%), alongside upregulation of SNAIL1 (+295%) and Endoglin (+116.5%), implicating modulation of angiogenesis and tumor progression pathways. The findings demonstrate that RocA possesses multiple anticancer mechanisms in TNBC cells, underscoring its potential for advancement into preclinical studies.