Infection of sheep by gastrointestinal nematodes (GIN) in pastoral systems is one of the main causes of economic loss and degradation of animal welfare. Treatments and control are based on the use of anthelmintics, eprinomectin (EPR) being the last and still efficient drug available in dairy farms without milk withdrawal period. However, loss of drug efficacy is threatening the success of GIN control. The efficacy of EPR was tested in dairy ewe farms in south of France when suspicions of loss of drug efficacy were raised by veterinarians, using fecal egg count reduction tests (FECRT). This test was reinforced by individual HPLC analysis of drug concentrations in the serum of treated ewes. The activity of EPR against Haemonchus contortus isolates from these farms was phenotypically monitored by measuring L3 larvae motility (WormMicroTracker® device). Lack of efficacy of EPR against H. contortus was confirmed by the low FECRT measured in several dairy sheep farms, while the EPR concentration in plasma of treated animals was sufficient for anthelmintic efficacy. When compared to matched susceptible isolates, the phenotypic evaluation of drug activity confirmed that H. contortus L3 larvae were resistant to drugs of the macrocyclic lactone family, including EPR. The presence of the pathogenic H. contortus in this important dairy region requires an urgent evaluation of the drug resistance status of the worm populations. By combining in vivo and in vitro tests, we provide a relevant approach for efficiently monitoring for anthelmintic resistance.