A total of 70 isolates were collected from neonates unit, Zagazig university hospital and identified to be coagulase-positive Staphylococcus aureus. The study was planned to evaluate and compare the methods of determination of minimum inhibitory concentrations for some antibiotics against S. aureus isolates. In disk diffusion method, the most effective drug was ciprofloxacın (77%) and the least effective was chloramphenicol (50%) The MIC range, MIC₅₀ and MIC₉₀ values by broth microdilution and agar dilution methods were closely related. The most effective drug against S.aureus isolates was ciprofloxacin then cefotaxime while, ampicillin/ sulbactam showed lower activity with higher MIC range, MIC ₅₀ and MIC₉₀ than expected E-test results showed high percentage of agreement with most of broth microdilution results and the difference was detected to be within the limit (±1-2 dilution). By using broth microdilution method as reference method and analysis of results by scattergram. Very major discrepancies were found in ciprofloxacin (3.5%) and cefotaxime (1.5%) Major discrepancy was noticed only with rifampicin (3%) and minor discrepancies were detected in all of antibiotics except ampicillin/ sulbactam which showed 100% agreement and there are no discrepancies The multidrug resistant isolates showed a plasmid DNA of sizes ranged from 8-30 kb with the transefer of ampicillin, cefotaxime, chloramphenicol and tetracycline resistance to the competent E. coli cells. In concolusion, E-test is considered efficient and: simple method for rapid determination of antibiotic MIC and activity.