This study aimed to characterize the different Staphylococci recovered from mastitic cows and buffaloes. A total of 126 mastitis milk samples were aseptically collected from clinically mastitic animals including 87 cows and 39 buffaloes. Bacteriological examination and biochemical identification using VITEK-2-compact-SYSTEM revealed that a total of 94 Staphylococcus isolates (74.6%) were recovered; 56 isolates (59.6%) and 38 isolates (40%) from cows and buffaloes, respectively. S. aureus was the most predominant isolate (n=26; 15 from cows and 11 from buffaloes) with a percentage of 27.7%. Moreover, 68 coagulase-negative staphylococci (CNS) isolates (72.3%) were identified of which; 21 S. epidermidis (22.3%); all isolates were from cattle, followed by 18 S. lentus (19.1%); 8 and 10 from cows and buffaloes, respectively, 17 S. simulans (18%); 6 and 11 isolates, respectively, and finally 12 S. hominis (12.9%); 5 and 7 isolates, respectively. Antimicrobial susceptibility testing showed that all isolates were sensitive to ceftriaxone, ciprofloxacin, ofloxacin and sulfamethoxazole-trimethoprim. On the contrary, all isolates were resistant to penicillin and streptomycin. Multidrug resistance (MDR) was detected in 21 (22.3%) Staphylococci isolates. Biofilm formation capacity was phenotypically assessed on YESCA CR agar medium and showed that all Staphylococci isolates were curli-producing. Application of PCR technique revealed that sed, seb genes were the most prevalent genes in all isolates, followed by fnbA gene which was detected in 80% of the isolates, and then mecA, blaZ, and icaA with percentages of 60%, 40%, and 40%, respectively.