Abstract Background Several species of nematophagous fungi exist in nature that can capture and kill nematodes as natural predators of soil-dwelling worms. These are important in agriculture and animal husbandry as biological control agents. The diversity of nematophagous fungi found from soil had not been studied in Ethiopia. Objective This study aimed to isolate Nematophagous Fungi from Soil Samples Collected From three Different Agro-Ecologies of Ethiopia. Methods Cross-sectional study was conducted and samples were collected from three different agro-climatic zones of Ethiopia; Debre-Berhan (highland), Bishoftu (mid-altitude), and Awash (lowland). Twenty-seven soil samples were randomly taken from each of the three different agro-ecological climates (9 from each agro-ecological climatic zone). For each study site, samples were collected from the soil of decomposed animal feces/dung, agricultural/farmlands, and forest lands in triplicates. Results The present study disclosed that nematophagous fungi were widespread from the study area. A total of 33 species of nematophagous fungi belonging to four genera, Arthrobotryes, Paecilomyces, Monacrosporium, and Harposporium were identified. Arthrobotrys were the most commonly isolated genera followed by Paecilomyces. The six identified species were Arthrobotrys oligospora, Paecilomyces lilacinus, Arthrobotryes dactyloides, Monacosporum eudermatum, Harposporium helicoides, and Monacosporum cionopagum. Conclusion This study indicated that Arthrobothryes oligospora was the most common species in Bishoftu and Awash whereas. In Debre-Berhan, Paecilomyces lilacinus was the most prevalent species. Monacosporum cionapagum was not isolated from dung soil and agricultural soil whereas Harposporium helicoides and Arthrobothryes dactyloides were not found from dung and forest soil respectively.