The lack of anthelmintic products licensed for donkeys and the rising number of small donkey milk farms in the countries of Western Europe and Italy have led to an increased interest in the study of reliable and safe plant-derived treatment alternatives. In this study, the aqueous extracts of Achillea millefolium L. (flowers), Artemisia absinthium L. (aerial parts), Centaurium erythraea Rafn. (flowers), Gentiana asclepiadea L. (rhizomes and roots), Inula helenium L. (rhizomes and roots) and Tanacetum vulgare L. (aerial parts), have been tested in vitro for their potential ovicidal and larvicidal activity against donkey nematodes. An egg-hatching assay (EHA) and larval development assay (LDA) were performed for the in vitro study, and median lethal concentration (LC-50) values for both EHA and LDA were calculated using probit analysis. All tested plant extracts showed strong anthelmintic activity against strongyle eggs and larvae at concentrations ranging between 125 and 1.95 mg/ml, except for C. erythraea, which exhibited very little or no effect at all at the tested concentrations. A strong ovicidal effect was observed in A. absinthium, with an LC-50 value of 0.486 mg/ml (95% confidence interval (CI) 0.21–1.09). Gentiana asclepiadea showed high efficacy against strongyle larvae, with an LC-50 value of 0.041 mg/ml (95% CI 0.01–0.16). The most significant (P < 0.01) anthelmintic activity was exhibited by I. helenium, with an LC-50 value of 0.041 mg/ml (95% CI 0.01–0.16) for EHA and 0.41 mg/ml (95% CI 0.27–0.62) for LDA. The results proved the anthelmintic efficacy of the tested plant extracts, highlighting the need for further research into plant bioactive molecules both in vitro and in vivo.