Increasing resistance towards anthelmintic drugs has necessitated the search for alternative treatments for the control of gastrointestinal nematode parasites. Animals fed on chicory (Cichorium intybus L.), a temperate (pasture) crop, have reduced parasite burdens, hence making C. intybus a potentially useful source for novel anthelmintic compounds or a diet-based preventive/therapeutic option. Here, we utilized in vitro bioassays with the parasitic nematode Ascaris suum and molecular networking techniques with five chicory cultivars to identify putative active compounds. Network analysis predicted sesquiterpene lactones (SL) as the most likely group of anthelmintic compounds. Further bioassay-guided fractionation supported these predictions, and isolation of pure compounds demonstrated that the SL 8-deoxylactucin (8-DOL) is the compound most strongly associated with anti-parasitic activity. Furthermore, we showed that 8-DOL acts in a synergistic combination with other SL to exert the anti-parasitic effects. Finally, we established that chicory-derived extracts also showed activity against two ruminant nematodes (Teladorsagia circumcincta and Cooperia oncophora) in in vitro assays. Collectively, our results confirm the anti-parasitic activity of chicory against a range of nematodes, and pave the way for targeted extraction of active compounds or selective breeding of specific cultivars to optimize its future use in human and veterinary medicine.