Discovery of drugs for the treatment of opisthorchiasis and schistosomiasis is a high priority. The basic metabolic cytochrome P450 (CYP) system in parasitic flatworms contains a single gene. CYP of the liver fluke Opisthorchis felineus, the causative agent of opisthorchiasis, is important for survival of the worm, so it may be a promising target for therapeutics against liver fluke infection. The aims of this study were: (i) to analyse in-vitro anthelmintic activity of various CYP inhibitors using standard motility and mortality assays against juvenile and adult O. felineus worms; and (ii) to characterize their anthelminthic effects. Azole inhibitors (ketoconazole, miconazole, triadimenol, clotrimazole and 4-phenyl imidazole) and other inhibitors of haem-containing enzymes (disulfiram, metyrapone, benzyl isothiocyanate, and ticlopidine) were tested. This study revealed that inhibitors of haem mono-oxygenase enzymes possess anthelmintic activity. The most effective anthelmintic agents against the newly excysted metacercariae (NEM) were the antifungal agents miconazole [concentration to reduce the response by 50% (IC50) 0.79 µM] and clotrimazole (IC50 1.25 µM), both approved by the US Food and Drug Administration. The activity of miconazole and clotrimazole was comparable to that for praziquantel (IC50 0.98 µM). In addition, 100% mortality was observed among NEM after 1 d of treatment with 10 µM miconazole, after 3 d of treatment with 10 µM clotrimazole, or after 7 d of treatment with 40 µM ketoconazole. When various CYP inhibitors were tested on adult worms, clotrimazole, miconazole and ketoconazole were found to be the most effective (IC50 13–20 µM). It is speculated that CYP may represent a promising drug target for combined treatment with other anthelmintic agents. The use of inhibitor–drug combinations may improve the action of standard anthelmintic agents.