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<i>Background and Objective</i>: <i>Staphylococcus epidermidis</i> is an opportunistic pathogen from pediatric bacteremia that is commonly isolated. Biofilm is the major virulence factor of <i>S. epidermidis</i>; however, the role of biofilm determinants in biofilm formation is highly contradictory and diverse. The current study aimed to investigate the role of polysaccharide-dependent and polysaccharide-independent pathogenic determinants in biofilm formation under physiological stress conditions. <i>Materials and Methods</i>: The isolates (<i>n</i> = 75) were identified and screened for the <i>icaADBC</i> operon, <i>IS256,</i> and an array of MSCRAMMs (Microbial Surface Component Recognizing Adhesive Matrix Molecules) through PCR analysis. The activity of the <i>icaADBC</i> operon was detected by Congo red assay, and the biofilm formation was analyzed through microtiter plate assay. <i>Results</i>: <i>S. epidermidis</i> isolates produced biofilm (<i>n</i> = 65; 86.6%) frequently. The <i>icaA</i> was the major representative module of the actively expressing <i>icaADBC</i> operon (<i>n</i> = 21; 80.7% sensitivity). The MSCRAMMs, including <i>fbe</i> (<i>n</i> = 59; 90.7%; <i>p</i> = 0.007), and <i>embp</i> (<i>n</i> = 57; 87.6%; <i>p</i> = 0.026), were highly prevalent and associated with biofilm positive <i>S. epidermidis</i>. The prevalence of <i>icaADBC</i> operon in biofilm positive and negative <i>S. epidermidis</i> was not significant (<i>n</i> = 41; 63%; <i>p</i> = 0.429). No significant association was found between <i>IS256</i> and actively complete <i>icaADBC</i> operon (<i>n</i> = 10; 47.6%; <i>p</i> = 0.294). In the presence of 5% human plasma and glucose stress, <i>S. epidermidis</i> produced a strong biofilm (<i>n</i> = 55; 84.6%). <i>Conclusion</i>: The polysaccharide-dependent biofilm formation is significantly replaced (<i>n</i> = 21; 28%; <i>p</i> = 0.149) by a polysaccharide-independent mechanism (<i>n</i> = 59; 90.7%; <i>p</i> = 0.007), in which the MSCRAMMs might actively play their role. The fibrinogen-binding protein and extracellular matrix-binding protein might be potential anti-biofilm drug targets, markers of rapid diagnosis, and potential vaccine candidates of <i>S. epidermidis</i> involved in pediatric bacteremia.