Mastitis is a costly and one of the most economically important production diseases affecting the dairy industry. A cross-sectional study was conducted from December 2015 to May 2016 in Adwa and Enticho Towns, Tigray, Ethiopia with the objectives of estimating the prevalence of bovine mastitis, isolation, characterization, and determination of antimicrobial profiles of E. coli and S. aureus from milk samples of dairy cows. A total of 156 lactating dairy cows from 23 smallholder dairy farms were sampled. Data related to herd size, breed, body condition, milking practice, farm hygiene, udder, and milk abnormalities were collected. California Mastitis Test (CMT) was performed. CMT-positive samples were collected. Bacteriological isolation, identification, and antibiogram testing were conducted. Data were recorded and analyzed for descriptive statistics. The overall prevalence rates of bovine mastitis, clinical mastitis, and subclinical mastitis in the study areas were found to be 35.9%, 3.85%, and 32%, respectively. Breed-based prevalence of mastitis was 21.74% and 41.8% for local and crossbreeds, respectively, (χ²=5.683; p ≤ 0.05). The overall prevalence of E. coli and S. aureus were 14.1% and 8.3%, respectively. The antimicrobial susceptibility test revealed that S. aureus was 100% susceptible to amoxicillin, kanamycin, and penicillin but 95.5% of E. coli isolates were susceptible to kanamycin, oxytetracycline, and streptomycin. Hence, regular CMT testing should be undertaken on every farm to screen subclinical mastitis-positive cows and all dairy farms should follow a good hygienic milking practice.