The nematocidal effect of Pleurotus ostreatus (white variety of oyster mushroom) aqueous extract (AE) was evaluated against Haemonchus contortus eggs and infective larvae (L3) in vitro and in artificially infected gerbils (Meriones unguiculatus). The chemical analyses indicated that constituents of AE are tridecanoic, tetradecanoic, linolelaidic, 9,15-octadecadienoic, and oxalic acids. P. ostreatus extract inhibited larval hatching by 100% at the concentration of 2.24 mg/mL and (50% effective concentration) EC50 of 0.73 mg/mL. In the larval development test, AE induced a larvicidal effect at the concentration of 50 mg/mL and EC50 of 17.24 mg/mL. The larval migration test revealed a reduction of 94.7% at a concentration of as low as 4 mg/mL and EC50 of 1.25 mg/mL. No significant effects of treatment with P. ostreatus AE were seen on H. contortus in the gerbil model. Thus, our results demonstrate an important nematocidal in vitro effect of P. ostreatus AE against the parasite H. contortus. However, further investigations are necessary to confirm the anthelmintic potential of P. ostreatus extract in small ruminants.