We recently identified a chromone derivative, 5-(4-bromobenzyloxy)-2-(2-(5-methoxyindolyl)ethyl-1-carbonyl)-4H-chromen-4-one, named here as chromone 1, as a potent, selective, nontoxic, and nontransported inhibitor of ABCG2-mediated drug efflux (Valdameri et al. J. Med. Chem. 2012, 55, 966). We have now synthesized a series of 14 derivatives to study the structure-activity relationships controlling both drug efflux and ATPase activity of ABCG2 and to elucidate their molecular mechanism of interaction and inhibition. It was found that the 4-bromobenzyloxy substituent at position 5 and the methoxyindole are important for both inhibition of mitoxantrone efflux and inhibition of basal ATPase activity. Quite interestingly, methylation of the central amide nitrogen strongly altered the high affinity for ABCG2 and the complete inhibition of mitoxantrone efflux and coupled ATPase activity. These results allowed the identification of a critical central inhibitory moiety of chromones that has never been investigated previously in any series of inhibitors.