In this study total of 416 milk samples (104 buffaloes) which were reared in a smallholder private cases hand milked in Sohag Governorate were examined. Clinical screening of examined animals beside the results of California mastitis test for apparentely healthy animals revealed that the overall prevalence rate of clinical and subclinical mastitis were 22.1% and 26.9%, respectively. The incidence of one quarter infection was the highest followed by all quarters in clinical and subclinical mastitis cases at the percentage of 30.4%, 35.7%, 26.2% and 28.6% respectively. The third lactating season was the highest incidence of both clinical and subclinical mastitis at the percentage of 28.6% and 35.3% respectively. By bacteriological examination 56 isolates from clinical mastitic cases and 68 isolates from subclinical mastitic animals were detected. Staphylococcus aureus , E.coli and , Streptococcus agalactia were isolated by ratio 16.1%, 33.9%, 14.3% and 29.4%, 22.1%,10.3 in cases of clinical mastitis and subclincal ones respectively. staphylococcus aureus and E. coli staphylococcus aureus and streptococcus agalactia and staphylococcus aureus and E-coli microorganisms were mixed isolated by ratio 7.1%, 7.1% and 5.4% in cases of clinical mastitis and 8.8%,4.4%, 4.4% in subclinical mastitic cases. Mixing of more than 3 isolates or more was the rate of clinical mastitis 8.9%, whereas subclinical mastitic were 8.8%. Simplex and multiplex PCRassays showed that E-coli isolates were the highest causative agents of clinical mastitis and staphylococcus aureus was the major cause of subclinical mastitic ones. Antimicrobial susceptibility testing revealed that all isolates were highly sensitive to Gentamycin Ceprofloxacin, Lincomycin and Novobiocin followed by Chloramphenicol, Cephalothin and Amoxycillin