Coagulase-negative staphylococcus (CNS) is the predominant group of bacteria in mastitis prevalence studies worldwide. An important virulence factor is the biofilm formation, once it assists in the persistence of the bacteria in the mammary epithelium. The aim of this study was to investigate the icaA, icaD, and bap genes in strains of CNS antimicrobial resistant isolated from bovine mastitis. Forty CNS (i.e., S. capitis, S. chromogenes, S. epidermidis, S. hominis, S. sciuri, S. simulans, and S. warneri), exhibiting a resistance profile (ampicillin, clindamycin, florfenicol, gentamicin, kanamycin, linezolid, neomycin, oxacillin, penicillin, streptomycin, trimethoprim-sulfamethoxazole and tetracycline, and mecA gene negative), isolated from cows and heifers with mastitis, from commercial farms in São Paulo State, Brazil, were used. The extraction of genomic DNA was performed using DNA Kit RTP Bacterium - Invitek. The genes amplification was performed by PCR and the reaction product was visualized on 2% agarose gel electrophoresis in 1× TBE buffer (1 M Tris base, 0.9 M boric acid, 0.01 M EDTA) stained 0.5% red gel. For positive control, they were used to S. aureus ATCC BAA-977 and S. xylosus DSM-20266. Descriptive statistical analysis was performed by calculating relative frequencies (PROC FREQ; SAS Institute, 2011) of resistant strains containing genes for biofilm production. Of the 40 isolates of CNS, 2.5% (n = 1) were positive for the icaA gene and 12.5% (n = 5) for the icaD gene, while were all negative for bap gene. The biofilm formation of staphylococcal is a complex process and involves the different mechanisms and gene regulation. The biofilms confer increased the capacity of adhesion and protection against the mechanisms of defense of the host and action of the antimicrobial agents, made difficult the elimination of the pathogen. Further studies are important for the understanding of the mechanisms of virulence and pathogenicity by strains of CNS, once these bacteria are frequently isolated in infections of the bovine mammary gland, representing the damages for the producers, the industry, and the public health.