Cyclooxygenases (COX) are enzymes responsible for metabolizing arachidonic acid into important inflammatory mediators, prostanoids. COX-2 is the inducible isoform and despite their efficacy, its available inhibitors present an increased risk of cardiovascular events, justifying the search for new molecules without this adverse effect. Chalcones are polyhydroxylated aromatic compounds with anti-inflammatory potential. However, their effect on COX-2 remains little explored. The main objective of this work was to evaluate the COX-2 inhibitory activity of the chalcones 2',3,4,4',6'-pentahydroxychalcone (5OH), 2',3,4,4',6'-pentamethoxychalcone (5OMe) and 2',3,4,4'-tetrahydroxychalcone (butein), at non-cytotoxic concentrations, in an experimental human whole blood model. The cytotoxicity of these chalcones was assessed through their effect on the viability of human blood cells, using UV/Vis spectrophotometry and flow cytometry techniques. The anti-inflammatory activity was evaluated through the quantification of prostaglandin E2 production, via COX-2, in human blood. The obtained results showed that none of the chalcones under study were cytotoxic. Butein was the only one that demonstrated a concentration-dependent inhibitory activity (40 ± 8%, for 50 µM). Under the present experimental conditions, 5OH and 5OMe showed no activity. In conclusion, butein seems to present some potential as COX-2 inhibitor. This chalcone may thus serve as a base structure for the development of new derivatives with anti-inflammatory activity.