The response of B16F10 melanoma and Melan-a melanocyte cell lines to treatment with five polyhydroxylated flavonoids and gallic acid, after 24 and 72 h of exposure, was determined, and the relationship between any antiproliferative effects observed and the chemical structure is discussed. After 24 h, none of the studied compounds showed significant cytotoxic activity in the B16F10 cell line, whereas compounds with an adjacent trihydroxylated substitution pattern did affect the viability of the Melan-a cell line. After 72 h of exposure, myricetin, baicalein and gallic acid significantly inhibited both B16F10 and Melan-a cell cultures, whereas luteolin and quercetin had only a moderate effect. Eriodictyol only had an effect on Melan-a cell viability, which was reduced slightly. These results suggest that the presence of a C2-C3 double bond and three adjacent hydroxyl groups in the flavonoid A- or B-rings confers greater antiproliferative activity to the flavonoid.