The aim of this study was to assess the changes in strongylid communities of domestic horses after two decades of regular anthelmintic treatments; the changes in prevalence and relative abundance of individual strongylid species and their contribution to the observed alterations were estimated. The study was conducted in 2023; data collected in 2004 were used for comparison; 39 horses from two farms in Ukraine (22 horses in 2004 and 17 in 2023) were examined. In total, 18,999 strongylid specimens were collected by in vivo diagnostic deworming method before (in 2004, 9119 specimens) and after (in 2023, 9880 specimens) frequent application of anthelmintics. Strongylids were identified morphologically. Thirteen strongylid species were found in horses in 2023; only small strongylids (Cyathostominae) were recorded. In 2004, 21 species were found: 6 species of large strongylids (Strongylinae) and 15 of cyathostomins. Species richness (Margalef’s index) and species diversity (Shannon’s and Simpson’s indexes) decreased over two decades on both farms; the Berger-Parker dominance index for C. nassatus dramatically increased up to 74.4. The dissimilarity of strongylid communities of 2023 and 2004 was primarily connected with the disappearance of large strongylids and rare cyathostomin species after two decades of regular anthelmintic treatments. SIMPER analysis revealed that C. nassatus and C. catinatum mostly contributed to this dissimilarity. A trend of gradual transformation of the strongylid community structure from multimodal (in 2004) to bimodal (in 2023) was observed on both farms.