Bovine pericardium a cellular material (BPAM), composed of decellularized collagen fibers, has been used in varied surgical procedures. Biomaterial-associated infections by human pathogens, during clinical procedures, are of significant medical concern. Biofilm formation by human pathogens has been increasingly described as a multifactorial and multistep process associated with infection of implanted medical devices. This study aimed to investigate the ability of biofilm formation on bovine pericardium patches (BPP), and the influence of virulence features of multi-drug resistance– MDR Staphylococcus haemolyticus blood isolates from neonate and adult hospitalized patients. The influence of phenotypic and genotypic virulence features in the ability of biofilm formation on BPP and abiotic surfaces were analyzed. Collagen-binding properties and ability of biofilm formation favored colonization, viability, and persistence in BPAM by S. haemolyticus strains. A higher adhesive activity, autoaggregation microcolonies, and ability of biofilm formation on BPP, independent of slime production were expressed by the invasive strain from neonate patients. The genes atl and fbp were detected for both S. haemolyticus strains with adhesive properties to collagen type I fibers and biofilm formation on BPAM. BPAM-biofilm producer S. haemolyticus strains also expressed the ability of biofilm production in the presence of Oxacillin, Vancomycin, and Linezolid antimicrobial agents, as shown on glass and polystyrene surfaces. Data indicate the involvement of multiple selective activities of virulence mechanism of varied nosocomial pathogens in contamination, persistence, and dissemination from biomaterials, through the bloodstream and tissues during human infections, such as MDR S. haemolyticus strains. Significance and Impact of Study: This work emphasized the possibility of contamination of BPP by human pathogens and the persistent infection following surgery of patients involving exposure to antimicrobial agents, including the ability of biofilm formation by S. haemolyticus on surfaces abiotic and biotic (BPAM).