Verbesina macrophylla is used in folk medicine to treat infections, inflammation and fever. This work aimed to evaluate the biological activity and phytochemistry of the species. Extracts in hexane, ethyl acetate and ethanol from the leaves of V. macrophylla revealed, using the microdilution method, activity against Gram positive bacteria (B. cereus, S. aureus, B. subtilis and M. luteus), with Inhibitory Concentration Minimum (MIC) between 500 µg/mL and 7.81 µg/mL. Fractionation of the extract in ethyl acetate resulted in 51 samples, all inactive for Gram-negatives. Four fractions showed activity against C. albicans and two against A. niger, at a concentration of 1000 µg/mL. Analysis of NMR data from fraction 19-20 revealed the presence of two isomers: (6-O-b-E-p-coumaroyl-4a-hydroxyeudesmane and 6-O-b-Z-p-coumaroyl-4a-hydroxyeudesmane), active for all Gram positive bacteria, bactericidal for B. cereus and S. aureus, with MIC between 2.5 and 3.9 µg/mL. The cytotoxicity test on red blood cells of the extracts showed slight hemolysis only for the hexane extract. The kinetic study of microbial growth of the Vm F19-20 fraction using the time-kill method revealed a dose-dependent effect for the B. subtilis strain.