Infections by gastrointestinal nematodes negatively affect small ruminant health and at the same time cause substantial economic losses worldwide. Because resistance to conventional anthelmintic compounds is growing, target studies evaluating the effectiveness of alternative ingredients of botanical origin on gastrointestinal nematodes are needed. In this study, we evaluated the in vitro anthelmintic activity of Ruta chalepensis L. extracts on the third-stage larvae of sheep gastrointestinal nematodes. A methanol extract showed the highest anthelmintic activity, with an EC50 = 0.10 ± 0.06 mg/ml after 96 h, while the essential oil had an EC50 = 1.45 ± 1.22 mg/ml after 48 h. Moreover, three secondary metabolites of the essential oil, i.e. 2-decanone, 2-nonanone and 2-undecanone, showed EC50 values of 0.07 ± 0.06, 0.25 ± 0.29 and 0.88 ± 0.73 mg/ml at 24 h, respectively. The present study indicated that the R. chalepensis methanol extract, the essential oil and its metabolites 2-decanone, 2-nonanone and 2-undecanone showed promising anthelmintic activity on gastrointestinal nematodes.