This study examined biofilm-producing Staphylococcus epidermidis isolates from Menoufia University Hospitals, Egypt, and the antibiofilm effects of AgNPs, chlorhexidine, and iodine. About 162 staphylococcal isolates from clinical samples were phenotypically identified as S. epidermidis using Vitek 2, with 71 (43.82%) verified. Tissue culture plate detection and conventional PCR genotyping for ica genes revealed biofilm-producing phenotypes. S. epidermidis isolates developed biofilms (94.36%, 67/71) when subjected to various antiseptic concentrations and durations, including chlorhexidine digluconate (0.025%, 0.035%, and 0.05%), povidone-iodine (3.5%, 7.5%, and 10%), and AgNPs (100, 75, 50, and 25 μg/ml). All biofilms were suppressed by chlorhexidine at 0.05% concentration and varying exposure durations. All povidone-iodine concentrations worked at 10 min; however, 3.5% was ineffective at 5 min. Only 10% concentration prevented biofilm growth at 1 min. AgNPs' antibiofilm impact is concentration-dependent, with the most effective concentration at 100 μg/ml (79.42%), followed by 75 μg/ml (74.91%), 50 μg/ml (70.71%), and 25 μg/ml (62.83%). Chlorhexidine was efficacious in vitro at a therapeutically available concentration of 0.05% and a short exposure duration of 1 min, but povidone-iodine required greater concentrations and longer exposure times. The study found that AgNPs have varying antibiofilm effects, with the most robust inhibition at 100 μg/ml concentration.