Bovine mastitis has a negative impact through economic losses in the dairy sector across the globe. A cross sectional study was carried out from September 2015 to July 2016 to determine the prevalence of bovine mastitis, associated risk factors and isolation of major causative bacteria in lactating dairy cows in selected districts of central highland of Ethiopia. A total of 304 lactating cows selected randomly from fi ve districts were screened by California Mastitis Test (CMT) for subclinical mastitis. Based on CMT result and clinical examination, over all prevalence of mastitis at cow level was 70.62% (214/304). Two hundred fourteen milk samples collected from CMT positive cows were cultured for isolation of major causative bacteria. From 214 milk samples, 187 were culture positive and the most prevalent isolates were Staphylococcus aureus 42.25% (79/187) followed by Streptococcus agalactiae 14.43% (27/187). Other bacterial isolates were included Coagulase Negative Staphylococcus species 12.83% (24/187), Streptococcus dysgalactiae 5.88% (11/187), Escherichia coli 13.38% (25/187) and Entrococcus feacalis 11.23% (21/187) were also isolated. Moreover, age, parity number, visible teat abnormalities, husbandry practice, barn fl oor status and milking hygiene were considered as risk factors for the occurrence of bovine mastitis and they were found signifi cantly associated with the occurrence of mastitis (p < 0.05). The fi ndings of this study warrants the need for strategic approach including dairy extension that focus on enhancing dairy farmers’ awareness and practice of hygienic milking, regular screening for subclinical mastitis, dry cow therapy and culling of chronically infected cows.