Helminth infections in general and digenetic trematodes in particular cause a huge economic loss globally to our livestock. Gigantocotyle explanatum is a digenetic amphistome that infects the bile ducts of water buffalo and are highly prevalent in tropical and sub-tropical countries. In the present study, effects of an organophosphate compound, Metrifonate (MF) in three doses, viz., 9.4 × 10-5 M (Dose I), 14.4 × 10-5 M (Dose II), and 19.4 × 10-5 M (Dose III), have been studied in vitro, on the motility and on some enzymatic and non-enzymatic oxidative stress indices in G. explanatum. The worm's motility and their non-enzymatic oxidative stress biomarkers like lipid peroxides measured as thiobarbituric acid-reactive substances (TBARS) and reduced glutathione (GSH) were disrupted significantly in a dose-dependent manner. However, the enzymatic oxidative stress biomarkers like glutathione-S-transferase (GST) and superoxide dismutase (SOD) were affected by MF treatment in a biphasic manner. Exposure to Dose I significantly stimulated the activities of both GST and SOD, whereas exposure to Doses II and III resulted into significant inhibition in a dose-dependent manner. Our findings suggest that MF has potential to be a strong and effective anthelmintic, however, further studies in vitro as well as in vivo are needed to explore further these observations and understand the exact mode of MF action in G. explanatum and other trematodes of veterinary economic importance.