This study sought to identify novel and nontoxic biofilm inhibitors from the Actinomycete library for attenuating biofilm formation by Staphylococcus epidermidis. After investigating the antibiofilm activities of spent media from 185 Actinomycete strains using two S. epidermidis strains (ATCC 35984 and a clinical strain 5-121-2) as target bacteria, three strains of tested Actinomycete (TRM 46200, TRM 41337, and TRM 46814) showed a significant inhibition against S. epidermidis biofilm formation without affecting the growth of planktonic cells. The characteristics of three strains of supernatants suggested that hydrophilic compound possibly extracellular peptides or proteins from these three strains, confer the biofilm reduction in S. epidermidis. An attempt was made to assess their effects on biofilm components and cell surface hydrophobicities in order to disclose acting mechanisms. The crude proteins from spent media of three strains degraded not only exopolysaccharides but also extracellular DNA in S. epidermidis biofilm. The active substances in crude proteins caused S. epidermidis cells to become less hydrophobic. Given these results, the metabolites from Actinomycete strains should keep further attention as potential antibiofilm agents against biofilm formation of S. epidermidis, even biofilm infections of the other bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus epidermidis infections are frequently associated with biofilms that are difficult to eradicate with conventional antibiotics. The new biofilm inhibitors from Actinomycete will have a great value in the prevention and treatment of dairy cow mastitis and other biofilm-related infections.