The flavones eupafolin (6-methoxy 5, 7, 3’, 4’ tetrahydroxyflavone) and hispidulin (6-methoxy - 5, 7, 4’ tryhydroxyflavone), were extracted from dry leaves of Eupatorium litoralle. Eupafolin differs from hispidulin because it has a catechol ring. Previous studies demonstrated their ability to decrease oxygen consumption of mitochondria due to inhibition of the enzymes of the respiratory chain and for hispidulin the inhibition of the amplitude and the velocity of mitochondrial energized swelling in its presence were also demonstrated. We now evaluate the effects of eupafolin on isolated mitochondria using energized and deenergized swelling experiments. Eupafolin (50 - 200 μmol.L^-1) inhibited deenergized swelling velocity by 30%. In the presence of eupafolin a dose dependent effect was observed, the inhibition of energized swelling occurring at 100, 90, 75, 65 and 35% at 200, 100, 75, 50 and 25 µmol.L^-1 respectively. Such an effect on energized swelling is in agreement with previous data on enzymes of the respiratory complexe as well as the inhibition of swelling driven by the K⁺ diffusion potential, suggesting it indirectly interferes with the properties of the mitochondrial membrane. In order to evaluate possible differences in cellular effects promoted by different substituent groups in the basic flavone structure, eupafolin, hispidulin and commercial flavone (10 – 200 μmol.L^-1) were applied to B16F10 melanoma cells. Cell viability was determined using the Trypan Blue test. Eupafolin, flavone and hispidulin promoted reduction in cell viability by 90, 40 and 30% respectively at 200 μmol.L^-1. The flavone did not affect cell viability (10 – 25 µmol.L^-1) but hispidulin and eupafolin were cytotoxic at all concentrations tested. These results indicate that the cytotoxic activity is dependent on the substituents in the basic flavone structure. The presence of catechol ring B plays an important role in the high activity against B16F10. These findings also suggest that inhibitions of the above mitochondrial parameters may be an underlying mechanism for cytotoxicity.