Simple SummaryThe ability ofS. aureusto attach to different surfaces and assist with biofilm formation is described in many types of environments, but mainly in the milk processing environment. The development of biofilm is one of the most significant virulence mechanisms involved in the adherence of staphylococcal strains to living or non-living surfaces. Recently, many studies have revealed that biofilms formed in milking equipment may be a source of ongoingS. aureuscontamination. The results of this study indicate thatS. aureuswas the most represented pathogen in milk and on surfaces. In the current study, the biofilm-producing ability of the reference strain and isolates ofS. aureusobtained from surfaces and milk was determined. In all strains, the counts necessary for biofilm formation (>5 Log10CFU/cm2) were detected, except for the reference strain. The ability to produce biofilm from isolates ofS. aureuswas higher in comparison with the reference strain during the first 3 h. In addition, significant differences between types of environmental contamination—occurrence ofS. aureuson the floor, teat cup, and cow restraints, and the frequency of mastitis caused byS. aureus(p< 0.05)—were confirmed, which potentially poses a serious risk of this pathogen persisting in the milking environment and during biofilm formation.AbstractThis study was conducted to evaluate the incidence of mastitis in 153 dairy cows and to evaluate the kinetics of adhesion of isolates obtained from surfaces and milk in comparison with the reference strain (RS), CCM 4223. The surfaces of the floor, teat cup, and cow restraints were aseptically swabbed in three replicates (n = 27). Of the total number of infected cows (n = 43), 11 samples were found to be positive forStaphylococcus aureus, 12 samples tested positive for non-aureus staphylococci, 6 samples tested positive forStreptococcusspp., and 11 samples tested positive for other bacteria (Escherichia coli, Pseudomonasspp.) or a mixed infection. The most represented pathogen in milk (11/43) and on surfaces (14/27) wasS. aureus.The kinetics of adhesion of the reference strain and isolates ofS. aureuson stainless steel surfaces were determined after 3, 6, 9, 12, 24, and 48 h, and 3, 6, 9, 12, and 15 days of incubation. All strains reached counts higher than 5 Log10CFU/cm2needed for biofilm formation, except RS (4.40 Log10CFU/cm2). The isolates ofS. aureusrevealed a higher capability to form biofilm in comparison with RS during the first 3 h (p< 0.001). Thus, there is a significant difference between the occurrence ofS. aureuson monitored surfaces—floor, teat cup, and cow restraints—and the frequency with which mastitis is caused byS. aureus(p< 0.05). This finding raises the possibility that if various surfaces are contaminated byS. aureus, it can result in the formation of biofilm, which is a significant virulence factor.