Water hyacinth poses serious socio-economic and environmental problems in Ethiopia. To integrate fungal pathogens into water hyacinth management, a survey was conducted in the Rift Valley of Ethiopia. Based on morphological characterization and DNA sequencing, 25 fungal species were identified that belong to nine genera. Alternaria tenuissima, A. alternata, Aspergillus niger, Phoma sp., Curvularia trifolii, Mucor fragilis, M. racemosus, A. fumigatus, Fusarium oxysporum, and F. equiseti were the most common fungi detected. However, their occurrence was influenced by water wave action, temperature, season, and altitude. Among the fungal pathogens, A. alternata, A. tenuissima, F. oxysporum, F. equiseti, and Neofisicoccum parvum were highly pathogenic to water hyacinth. Alternaria alternata and A. tenuissima did not cause disease symptoms on ecologically important plant species (e.g. Noug, Tef, and Coffee). Application of the fungal pathogens on water hyacinth plants also showed 11%–67%, 22%–72%, 15%–55%, and 12%–50% reduction in fresh weight, dry weight, plant height, and root length of water hyacinth, respectively. This study suggests that fungal species have the potential to control water hyacinth biologically and provides baseline data for biological control efforts in the future.