In this study we isolated and characterized Staphylococcus xylosus, a coagulase-negative staphylococcal species considered as commensal and one of the prevalent staphylococcal species found in poultry bioaerosol. Isolates were obtained using air samplers and selective phenylethyl alcohol agar for gram-positive bacteria during 35-d periods at different times of the day. A total of 200 colonies were recovered and after basic biochemical tests were performed, presumptive staphylococci were subsequently identified by API Staph strips. A total of 153 (76.5%) staphylococci were found, among which 84 were S. xylosus (46 and 38 isolated inside and outside, respectively). Biofilm formation was observed in 86.9% of S. xylosus isolates, whereas 79.8% of them showed hemolytic activity. There was a strong correlation (92.5%) between biofilm formation and hemolytic activity. All 84 S. xylosus isolates were susceptible to amikacin, ampicillin/sulbactam, chloramphenicol, ciprofloxacin, gentamycin, kanamycin, linezolid, trimethoprim/sulfamethoxazole, and vancomycin. Resistance to nalidixic acid (86.9%), novobiocin (85.7%), penicillin (70.2%), lincomycin (46.4%), oxacillin (42.9%), ampicillin (27.4%), tetracycline (21.4%), erythromycin (11.9%), bacitracin (10.7%), and streptomycin (2.4%) was observed among the isolates. Resistance to tetracycline, lincomycin, erythromycin, and β-lactam antibiotics was occasionally linked to the tetK, linA, ermB, and blaZ genes, respectively. Random amplification of polymorphic DNA results showed similarity of 15 to 99% between isolates collected outside and inside the barn, indicating genetic diversity of these isolates. Our study indicates that characterization of poultry bioaerosol coagulase-negative staphylococcal species such as S. xylosus is necessary for assessing their safety status for both poultry and humans.