Persistent buffalo mastitis caused by Staphylococcus spp. gives rise to economic losses and may be resistant to antimicrobial therapy. The aim of the present study was to determine resistance patterns and the presence of mecA, blaZ, and efflux pump in Staphylococcus spp. isolated from cases of mastitis in Brazilian buffalo herds. Susceptibility to antimicrobials was determined by the disk diffusion test and detection of the mecA and blaZ genes by polymerase chain reaction. The efflux pump screening test was performed by growing samples in Muller-Hinton agar containing ethidium bromide. The percentages for resistance to the drugs tested were: 71.8% to penicillin, 49.2% to amoxicillin, 65.8% to oxacillin, 62.3% to cefquinome, 44.7% to cephalonium, 45.2% to ciprofloxacin, 32.6% to enrofloxacin, 58.7% to erythromycin, 42.7% to florfenicol, 34.6% to gentamicin, 35.1% to trimethoprim-sulfamethoxazole, 8.5% to tetracycline + neomycin + bacitracin, 43.2% to cephalothin, 38.1% to streptomycin, 58.7% to tetracycline, 31.6% to norfloxacin, 45.2% to ceftriaxone, 43.2% to nitrofurantoin, 57.7% to doxycycline, and 53.7% to cephalexin. Simultaneous resistance to 4 or more antimicrobial drug groups was observed in 112 isolates, using the mecA (11) and blaZ (79) genes, and efflux pump (47). It is concluded that Staphylococcus spp. isolates from cases of mastitis in Brazilian buffalo show varying levels of resistance to antibiotics, and caution should be exercised in choosing therapeutics in order to minimize the risk to public health.