Ethnobotanical study of medicinal plants in Negele Arsi district was conducted to document medicinal plants and associated indigenous knowledge. Ethnobotanical data were obtained using semi-structured interviews and focus group discussion from July to August 2020. Data were analyzed by descriptive statistics using Microsoft Excel, Stata and Statistical Package for Social Science.82 medicinal plant species belonging to 77 genera and 55 families were identified and documented. The highest number of medicinal plants was used in Aga (highland) study site (43 species). Out of total medicinal plants, 42(51.2%) species were reported to treat human aliments. Most of the medicinal plants were herbs (36.59%), followed by trees (31.71%). The leaf (37.80%) parts of medicinal plants were frequently used, followed by bark (20.73%). The most widely used mode of preparation in study area was crushing (50.00%) followed by powdering (23.17%). The most commonly used route of administration was oral (43.75%) followed by dermal (29.46%). Most of the medicinal plants were reported to be used in fresh (58.54%) condition to treat diseases. A Pearson correlation test indicated a positive and significant difference between age of informants and number of medicinal plant species cited and negative and significant difference between level of education and number of medicinal plant species cited. The t-test value indicates that there was significant difference between male and female informants in their knowledge of medicinal plants, and there was significant different between number of medicinal plants reported by key informants and household respondents. The highest proportion of informants (36.59%) cited Ocimum lamiifolium species and, Anemia disease category scored the highest number informant consensus factor (0.82) value. Preference ranking indicates that Ocimum lamiifolium ranked first in treating febrile illness human disease and Croton macrostachyus ranked first in treating blackleg livestock disease in the study area. Pair wise comparison indicated that Rotrariacristata ranked first to treat spider poison disease. Direct matrix ranking revealed that Cordia africana ranked first as a multipurpose medicinal plant. Agricultural expansion (41.96%) stood first as a factor threatening medicinal plants, followed by fuelwood collection (33.04%). To support local people effort on in-situ conservation and ex-situ conservation, further researchis needed to identify and conserve of medicinal plants in the study area.