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Aims: Eupatorium laevigatum Lam. is commonly used as anti-inflammatory, antiseptic, antirheumatic, and in the treatment of colds and ulcers. The present study aimed to characterize the active fractions of the aerial parts of E. laevigatum, isolate its major constituents and to evaluate its cytotoxic effects against human tumor cells. Methodology: Phytochemical analysis of the aerial parts of E. laevigatum detected the presence of flavonoids, saponins and coumarins. Nuclear magnetic resonance with carbon and hydrogen determined that coumarin to be scopoletin. The human cancer cell lines HT-29, NCI-H460, MCF-7 and RXF-393 were used to evaluate cytotoxicity through the sulforodamine B assay as well the evaluation of oxidative damage through the thiobarbituric acid reactive species assay. Short Research ArticleResults: Our study has shown that E. laevigatum crude extract and chloroform, ethyl acetate and butanol fractions are not cytotoxic in the concentrations used (up to 100 µg/mL), but the coumarin scopoletin isolated from the aerial parts of E. laevigatum presented a cytotoxic effect against NCI-H460 and RXF-393 cells (IC 50 value of 19.1 and 23.3 µg/mL, respectively). Scopoletin did not show any oxidative effect. Conclusion:The coumarin scopoletin can be found in E. laevigatum and this compound induces cytotoxicity in NCI-H460 and RXF-393 cell lines. Moreover, it is suggested that the cytotoxic effect of scopoletin is no related to oxidative damage.