Staphylococcus species are the most precarious micro-organisms in poultry meat, because they cause health problems to consumers and the economy of the country. Therefore, the current study was aimed to investigate the incidence rate of Staphylococcus species isolated from broiler chicken and hospitalized patient, detection the antimicrobial susceptibility of these isolates by using Vitek2 system and confirmation the presence of genes encoding for pathogenicity and antimicrobial resistance in detected isolates by PCR. To achieve these 200 samples were collected from poultry farms and hospitalized patient (100 for each) in dissimilar districts in Assiut province, to be subjected to bacteriological examination. The results showed that the incidence of Staphylococcus species was 35% and 45% in poultry and human samples respectively on mannitol agar. Vitek2 system divided poultry isolates to 11 isolates as coagulase-positive Staphylococci (CoPS) and 24 isolates as coagulase-negative staphylococci (CoNS). Whereas 20 human isolates alienated as CoPS and 25 isolates assigned as CoNS. Antibiogram cleared that 45.7% and 53.3% of Staphylococcus isolates from poultry and human were identified as methicillin-resistant Staphylococci respectively, also Staphylococcal spp. cleared a resistance to different types of antimicrobials such as penicillin, tetracycline clindamycin. Vitek2 system showed a significant ability to differentiate among Staphylococcus species and distinguish its antimicrobial susceptibility which was complicated by conventional method. PCR results showed that the Staphylococcal isolates of poultry and human were harboring genes encoding for pathogenicity (coa, hld and pvl) and antimicrobial resistance (mecA, vanA, cfr and BlaZ) while none of the isolates harbored sei and seh genes.