Bovine mastitis is a serious problem in the dairy herds all over the world. In this study 105 mastitic milk samples were collected from small holder's cows for bacteriological and molecular diagnosis. The prevalence of clinical and subclinical mastitis were 20.5% and 32% respectively which were detected by clinical examination and California Mastitis Test respectively. Bacteriological results revealed that Staphylococcus aureus was the most common isolated bacteria from both clinical and subclinical mastitis. Streptococcus species, Pseudomonas species, E. coli and Enterobacter species were also isolated. Application of multiplex PCR was effective in identification of bacteria causing mastitis directly from milk samples and from extracted DNA of bacteria