The overexpression of P-glycoprotein (P-gp) by tumor cells results in multidrug resistance (MDR) to structurally unrelated anticancer drugs. Combined therapy with MDR-related cytotoxins and MDR modulators is a promising strategy to overcome clinical MDR. This study was designed to screen potent MDR modulators from imidazole derivatives. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The intracellular accumulation of doxorubicin (Dox) was detected by fluorescence spectrophotometry. The function of P-gp was examined by Rhodamine 123 accumulation detected with flow cytometry (FCM). Among imidazole derivatives, FG020326, FG020327, and FG020318 were found to possess three- to fourfold stronger reversal MDR activity than verapamil, a well-known positive MDR modulator. Imidazole derivatives significantly increased the Dox accumulation and inhibited P-gp function exhibited by the increase of Rhodamine accumulation in MDR cells. The fold reversal of MDR was relative with the increase of Rhodamine accumulation. FG020326, FG020327, and FG020318 showed potent MDR reversal activity in vitro. Their mechanism of MDR reversal is associated with the inhibition of P-gp function and the increase of anticancer accumulation. These results suggest FG020326, FG020327, and FG020318 are promising to further study and develop.