A cross-sectional study was conducted from October 2007 to April 2008 on Holstein and Holstein-Zebu cross breds lactating dairy cows in and around Mekele to determine the prevalence, major risk factors and major bacterial pathogens of bovine mastitis in the study area. Simple random sampling of dairy herds, clinical examination of udder and milk, White Side Test, and bacteriological culture were used. Based on clinical examination of udder, milk, and white side tests, prevalence of mastitis was found to be 61.5% (4.0% clinical and 57.5% sub clinical cases). The overall prevalence of mastitis was significantly affected by age (χ2 = 22, P=0.000), breed (χ2 = 7.9, P= 0.006), teat condition (with or without lesion) (χ2 = 5.3, P= 0.025), parity (χ2 =8.2, P=0.017) and sanitation status of the dairy environment and associated practices (χ2 =19.0, P=0.000). Coagulase negative staphylococcus (50.2%), Staphylococcus aureus (22.9%), streptococcusagalactia (16.3%), streptococcus dysagalactia (7.3%) and E. coli (3.3%) were the major bacterial species isolated. In conclusion culling of old aged cows, improving the overall sanitation of farm, screening, early detection and treatment of mastitis, tick control and proper management of teat/udder injuries are recommended to combat the problem in the area.