Background: Staphylococcus epidermidis (S. epidermidis) has emerged as a major etiological factor in implant-related infections, primarily due to its biofilm forming ability. These biofilms enhances bacterial resistance to antimicrobial agents. Aim: This study aimed to detect and characterize drug-resistant S. epidermidis strains in clinical samples obtained from Menoufia University Hospitals, with evaluating their biofilm-forming ability and assessing the antimicrobial activity of chlorhexidine and povidone iodine. Methodology: Drug-resistant S. epidermidis strains were identified using the Vitek2 system and their biofilm producing ability was determined. The presence of biofilm-related genes (icaA and icaD) was confirmed through polymerase chain reaction (PCR). Multi-drug resistant S. epidermidis isolates capable of biofilm formation were exposed to varying concentrations of chlorhexidine (0.025%, 0.035%, 0.05%, 0.12%) and povidone iodine (1.5%, 3.5%, 7.5%, 10%) for different exposure times. Results: Of 354 clinical isolates, 75 (21.2%) were identified as drug-resistant S. epidermidis using Vitek2 system. Biofilm production was observed and confirmed by the presence of icaA / icaD genes among 61-isolates. Chlorhexidine demonstrated significant effectiveness in vitro at concentration of 0.05% with a short exposure time of 1 minute. In contrast, povidone iodine required higher concentrations and prolonged exposure times to exhibit antibacterial activity. Conclusion: Chlorhexidine was an effective antimicrobial agent against S. epidermidis, particularly when used at clinically available concentrations (0.05%) with short exposure time, whereas povidone-iodine required higher concentrations with prolonged exposure times. Further investigations are warranted to optimize the use of these antiseptics.