The aim of present study was to determine the occurrence and etiology of subclinical mastitis in buffaloes submitted to an automatic milking system. A total of 146 quarter milk samples from buffaloes without clinical mastitis were analyzed. Milk collection was performed before starting the morning milking. A single milk sample was obtained from one mammary quarter of each animal. The aseptic collection of samples for microbiological analysis was performed directly from the teat, disinfected with alcohol 70% (wt/vol). The milk was stored in a 15 mL sterile polypropylene tube, frozen and sent to the laboratory to isolate and identify bacterial organisms. The samples were incubated at 37°C for 8 h for the pre-enrichment stage, and subsequently plated on sheep blood agar, brain heart infusion agar and Sabouraud agar with chloramphenicol. After incubation, morphological characteristics of the isolated colonies and individual innocuous were evaluated, then plated in selective and differential media to observe the characteristic phenotypic aspects of the genera. A total of 140 (96%) samples were culture positive, and among these, 91 (65%) mammary quarters were infected by individual pathogens. From these, 47% were Corynebacterium spp., 25% were Staphylococcus spp., 13% Streptococcus spp., 7% Bacillus spp., 2% Klebsiella spp., 2% E. coli, 2% yeast and 1% Micrococcus spp. Corynebacterium bovis was the predominant mastitogenic organism, followed by Staphylococcus chromogenes. Among streptococci, Streptococcus agalactiae was the predominant organism. The mixed infections were detected in 35% quarters in different combinations. The most common combination was Staphylococcus spp. and Corynebacterium spp., followed by Streptococcus spp. and Corynebacterium spp., while S. aureus, S. equorum, Pseudomonas spp., Enterococcus spp. and Aerococcus viridans were present in mixed infections with low prevalence. Buffaloes managed in intensive farms may present subclinical mastitis cases. Therefore, attention is necessary in the drying management and other mastitis prevention practices, especially when the buffaloes are submitted to an automatic milking system.