Biofilm is one of the important virulence factors of staphylococci that plays a role in many device-related infections such as native valve endocarditis, otitis media, urinary tract infections, cystic fibrosis, acute septic arthritis, etc. Biofilm is a microbially derived sessile community of microorganisms, developed either from single or multiple microorganisms. Formation of biofilm is a two-step process: adherence of cells to a surface and accumulation of cells to form multilayered cell clusters. A trademark of biofilm formation in staphylococci is the production of polysaccharide intercellular adhesin. In the formation and regulation of biofilm, some biosynthetic genes (icaADBC) and some regulatory genes (icaR, sar, agr, rbf, sigma(B)) are involved. In this article, we reviewed the structure and formation of staphylococcal biofilm and its role in medical infections.