Biofilms are a constant concern in the food industry; understanding the effect of environmental conditions on biofilm formation is essential to develop effective control strategies. Therefore, this study was conducted to investigate biofilms formation by Staphylococcus epidermidis under various conditions. Biofilms were cultured in nutrient broth containing different carbon source concentrations (0–10 mg/mL) on polystyrene surfaces for 32 h of incubation at 37 ◦C or 55 ◦C, with quantification and enumeration at 8, 16, 24 and 32 h. S. epidermidis developed biofilms under all tested conditions; achieved the highest yield of biofilm biomass at 2.5 mg/mL for all carbon sources at 37 ◦C. The highest efficiency of extracellular polymeric substance (EPS) molecule production occurred under glucose availability in the growth environment, with a higher yield of biomass and a significantly smaller number of metabolically active cells than under other tested conditions. A condensed ball-shaped structure was observed under the lactose condition. Meanwhile, biofilms in the presence of maltose showed mainly opaque thick rich colonies, while a compact multilayered-shaped structure was exhibited under both glucose and sucrose conditions. These results contribute to a better understanding of the biofilm formation by S. epidermidis in order to reduce contamination and recontamination in the food industry.