Increasing resistance to anthelmintic drugs has necessitated the search for alternative treatments to control gastrointestinal nematode parasites. Animals fed Cichorium intybus L, a temperate (pasture) crop, reduce their parasite burden, making C. intybus a potentially useful source for new anthelmintic compounds or a diet-based prophylactic-therapeutic option. Here, we used in vitro bioassays with the parasitic nematode Ascaris suum and molecular network methods with five species of yeast to identify putative active compounds. Network analysis predicted sesquiterpene lactones (SL) as the most likely group of anthelmintic compounds. Subsequent biomass-based fractionation supported these predictions, and isolation of pure compounds showed that SL 8-deoxylactusin (8-DOL) was the compound most strongly associated with antiparasitic activity. In addition, 8-DOL was observed to have an anti-parasitic effect in synergistic combination with other SL. Finally, it was found that extracts obtained from the mushroom showed activity against two ruminant nematodes (Teladorsagia circumcincta and Cooperia oncophora) in in vitro assays. Overall, our results confirm the antiparasitic activity of the mushroom against a range of nematodes and pave the way for targeted isolation of active compounds or selective breeding of specific cultivars to optimize its future use in human and veterinary medicine.