Background: Staphylococci are implicated as the main causes of biofilm-associated neonatal infections (NIs). Objectives: This study aimed to investigate biofilm formation by Staphylococci isolated from newborns with neonatal sepsis and comparing and analyzing Staphylococcal biofilm-forming capacity and multidrug resistance. Methodology: One hundred blood cultures and thirty endotracheal tube specimens of neonates with septicemia were comprised in this study. Staphylococcal isolates were detected by conventional methods and investigated for biofilm formation by Microtiter plate (MTP), Tube method (TM) and Congo red Agar (CRA) detection methods followed by detection of icaA & icaD adhesin genes by Multiplex-PCR. Results: Among the isolated strains, fifty Staphylococcal strains were studied for biofilm formation and antibiotic susceptibility. They were 56%, 68%, 88% positive biofilm formers by MTP, TM and CRA detection methods, respectively. While, they were 28 (56%) positive biofilm formers by PCR. Out of the 28 icaAD positive staphylococcal isolates 27 (96.4%) were TM and CRA-positive, but only 15 (53.5%) were MTP-positive. The correlation between the sensitivity and specificity of phenotypic biofilm detection methods with PCR, was 96.4%, 96.4% and 53.5% sensitivity for TM, CRA and MTP methods, while they were 68.1%, 22.7%, 40.9% specificity comparing with PCR, respectively. There was a statistical significant difference between antibiotic resistances among them whereas strong biofilm formers were more resistant to all tested antibiotics compared to moderate and weak biofilm formers. Conclusion: TM is easy, reliable with excellent sensitivity method for detection of biofilm in neonatal nosocomial infections, as compared to CRA and MTP methods. Further investigation of the genetic variations of adhesion genes are needed for better evaluation of staphylococci biofilm forming capacity and multidrug resistance.