This study was carried out on a total of 550 lactating animals; 310 and 240 cows and buffaloes, respectively which were examined for signs of clinical mastitis (swelling, hotness, redness, and apparent milk change) from different dairy farms and veterinary units located at El-Fayoum Governorate during the period from May 2017 to November 2017. Clinical examination proved that out of these animals, a total of 126 animals (87 cattle and 39 buffaloes) were found with clinical mastitis. Streptococcus species were recovered from 73 animals including; 29(39.7%) and 44(60.0%) cows and buffaloes, respectively. Furthermore, out of the 73 Streptococci isolates recovered from cows and buffaloes; there were 10(13.7%) and 15(20.5%) S. agalactiae, 5(6.8%) and 10(23.7%) S. dysgalactiae, 8 (10.6%) and 7 (13.7%) S. uberis, 3(4.1%) and 10(13.7 %) E. fecalis and 3(4.1%) and 2(2.7%) S. lactarius, respectively. Anti-microbial susceptibility testing showed that the highest resistance was recorded against penicillin, gentamicin, streptomycin, and doxycycline (100%). Conversely, the highest sensitivity was recorded against ceftriaxone, ciprofloxacin, and sulfamethoxazole-trimethoprim (100%). Biofilm formation capacity was phenotypically assessed on YESCA CR agar medium and showed that all examined S. agalactiae and S. dysgalactiae were strong biofilm producers, meanwhile, 78%, 50%, and 75% of S. uberis, S. lactarius, and E. fecalis were biofilm positive isolates respectively. Application of PCR technique revealed that enterotoxins producing genes; sed, seb were found in 20% and 80% of isolates, in order. Biofilm-associated genes; fnbA and icaA genes were detected in 90% and 70%, respectively. Resistance genes; mecA and blaZ, genes were possessed in 90% and 70% of isolates, respectively.