Aim The objective of this study was to investigate the biofilm inhibitory activity of Streptomyces-derived actinomycin D against biofilm formation by Staphylococcus epidermidis. Methods and Results The microtitre plate method and microscopy were used to detect the biofilm formation of S. epidermidis. And an attempt was made to detect the effect of actinomycin D on important biofilm components, exopolysaccharides (EPS) in S. epidermidis using precolumn derivation HPLC. Also cell surface hydrophobicities of S. epidermidis were assessed to explore action mechanisms. The qPCR was performed to demonstrate the genetic mechanisms of biofilm formation by S. epidermidis. Unlike other antibiotics, actinomycin D (1 center dot 5 mu g ml(-1)) from Streptomyces luteus significantly inhibited biofilm formation by S. epidermidis. Additionally, it effectively inhibited S. epidermidis cells from adhering to glass slides. Actinomycin D downregulated ica locus and then the reduced polysaccharide intercellular adhesin production caused S. epidermidis cells to become less hydrophobic, thus supporting its anti-biofilm effect. Conclusion Streptomyces-derived actinomycin D is active in inhibiting the biofilm formation of S. epidermidis. Significance and Impact of the Study Actinomycin D can be used as a promising antibiofilm agent in inhibiting S. epidermidis biofilm formation. The study is also the first insight into how actinomycin D inhibited the biofilm formation of S. epidermidis. Actinomycin D could potentially be used to reduce the risk of biofilm-associated infections. Our study also suggests that the metabolites from Actinomycete strains keep further attention as potential antibiofilm agents against biofilm formation of S. epidermidis, even biofilm infections of the other bacteria.