Cooperia punctatais one of the most prevalent gastrointestinal nematodes affecting cattle under grazing conditions, and the increasing reports of anthelmintic resistance forces researchers to look for novel control measures. Previous reports have proposed the use of polyphenolic compound (PC) combinations (Coumarin:Quercetin (CuQ) and Caffeic-acid:Rutin (CaR)) against free-living stages (L3) ofC. punctata. The objective of this study was to assess the in vitro motility inhibition ofC. punctataadult worms and infective larvae using the Larval Motility Inhibition Assay (LMIA) and Adult Motility Inhibition Assay (AMIA), and to assess the structural and ultrastructural changes induced by both treatments using Scanning and Transmission Electron Microscopy. For the LMIA, infective larvae were incubated for 3 h in 0.8 mg mL−1and 0.84 mg mL−1of CuQ and CaR, respectively. For AMIA, six concentrations and five incubation periods (2, 4, 6, 12 and 24 h) were assessed using each PC combination.Cooperia punctatamotility was calculated as a percentage and corrected using control motility percentages. A multiple comparisons Brown–Forsythe and Welch ANOVA test was used to compare larval motility; and to fit the dose–response in AMIA, data were analyzed with a non-linear regression four-parameter logistic equation with a variable slope, using the computer program GraphPad Prism®V.9.2.0. Although larval motility was barely affected by both treatments (p> 0.05), adult worm motility was inhibited 100% and 86.9% after 24 h incubation with CuQ and CaR, respectively (p< 0.05). The best fit EC50for adult worm motility inhibition were 0.073 ± 0.071 mg mL−1and 0.051 ± 0.164 mg mL−1for CuQ and CaR, respectively. Main structural and ultrastructural lesions observed in both biological stages were: (i) L3sheath–cuticle complex disruption, (ii) collagen fibers degradation; (iii) hypodermic detachment, (iv) seam cell apoptosis and (v) mitochondrial swelling. The alterations observed suggest that the PC combinations interfere with the anatomy and physiology of the locomotive apparatus of the nematodes.