Objective: To isolate the chemical constituents of the aerial parts from Eupatorium illitum, elucidate their structures and evaluate their antiproliferative activity on human cancer cell lines. Materials and Methods: The ethanolic extract of Eupatorium illitum afforded five compounds, which were characterized using spectroscopic techniques and by comparison with data from the literature. Antiproliferative activities of selected isolates were evaluated. Results: The flavonoids Kumatakenin (1), Ermanin (2), 7-methoxy-aromadendrin (3) and Naringenin (4), together with 4-hydroxybenzoic acid (5) were isolated. Compounds 1, 2, 4 and 5 were evaluated for their antiproliferative activity on the human cancer cell lines A549 (lung), HBL-100 (breast), HeLa (cervix), SW1573 (lung), and T-47D (breast) presenting a wide range of bioactivities. In general, best results were observed for 5. Conclusion: Compounds 1-5 are reported for first time from Eupatorium illitum. Isolated phytochemicals show moderate to low antiproliferative activities when evaluated on the aforementioned human cancer cell lines.