Increase in antibiotic resistance in Staphylococcus aureus isolated from ear infection is a serious public health problem. The objective of this investigation was to determine the antibacterial resistance profile and genetic variability of the S. aureus isolated from adult patients with otitis externa (OE) and otitis media (OM) infections, Tehran- Iran. The disk diffusion was employed to detect the susceptibility of 45 S. aureus strains. Biofilm production was evaluated by microtiter plate assay. Genetic diversity of the isolates was determined by staphylococcal cassette SCCmec, spa, and MLST techniques. Resistance to mupirocin and vancomycin were identified in 40 and 2.2% of isolates. Out of the 45 S. aureus isolates, 41 (91.2%) strains were considered as positive biofilm strains at different levels. According to our results, S. aureus isolated from OM (44.4%, 20/45) were including CC8/ST239-SCCmecIII corresponded to spa types t860, t030, t037, t234, t421 (70%, 14/20) and CC/ST30-SCCmecIV corresponded to spa types t605 and t019 (30%, 6/20) while S. aureus isolated from OE (55.6%, 25/45) were including CC/ ST30-SCCmecIV corresponded to spa types t605, t345 and t1130 (52%, 13/25), CC/ST22-SCCmecIV corresponded to spa type t790 (20%, 5/25), CC8/ST8-SCCmecIV corresponded to spa type t008 (16%, 4/25), and CC/ST45-SCCmecIV corresponded to spa types t004 and t038 (12%, 3/25). This study highlighted genetic variability and strong biofilm formation ability among our isolates revealing its crucial role in enhancing the resistance of this bacteria to drugs. Thus, it is necessary to continue the epidemiological analysis to improve the control of ear infections related to S. aureus.  Kulcsszavak: Staphylococcus aureus, biofilm formation, otitis externa, otitis media, SCCmec, antibiotic resistance, multilocus sequence typing, spa