<p>Triple-negative breast cancer (TNBC) is an aggressive subtype with limited therapeutic options, underscoring the need for novel antitumor agents and more physiologically relevant in vitro models. Although species of the <i>Ageratum</i> genus exhibit biological activity, the antitumor potential of <i>Ageratum fastigiatum</i> and its evaluation in three-dimensional (3D) microenvironment have not been previously investigated. This study evaluated the cytotoxic, antiproliferative, and antimigratory effects of the ethanolic extract of <i>Ageratum. fastigiatum</i> on TNBC cells MDA-MB-231 using conventional two-dimensional (2D) cultures and a 3D tumor-on-a-chip model. In 2D cultures, cell viability assessed by the MTT assay revealed an IC₅₀ of 308&#xa0;µg/mL, and approximately 80% reduction in viability at a concentration of 500&#xa0;µg/mL. The extract significantly inhibited cell migration in scratch assays and reduced colony formation in clonogenic assays. Cell cycle analysis showed an increased sub-G0/G1 population at the IC₅₀ concentration, indicating apoptosis-associated cell cycle disruption. In the 3D microfluidic model, tumor cells embedded in a collagen hydrogel exhibited reduced sensitivity to the extract compared to 2D cultures, with viable cell areas of approximately 15%, compared to 20% under control group , highlighting the influence of the extracellular matrix on treatment response. Additionally, the extract inhibited cell migration by approximately 59% in the 3D model at the IC₂₅ concentration (289&#xa0;µg/mL). In conclusion, the ethanolic extract of <i>A. fastigiatum</i> demonstrated antitumor activity against TNBC cells, and the incorporation of 3D microfluidic models highlights the importance of tumor microenvironment based platforms for a more physiologically relevant evaluation of therapeutic candidates.</p>