Three pigmented secondary metabolites tetracenomycin D (1), resistomycin (2), and resistoflavin (3) were isolated from Streptomyces sp. EG1 and their antibiofilm activities were evaluated against Staphylococcus aureus and Escherichia coli. The isolated compounds were purified by different chromatographic methods, including Silica gel and Sephadex LH-20, and their chemical structures were elucidated by mass and NMR spectroscopy. Tetracenomycin D (1) inhibited 75% of both pathogens from creating biofilm at 400 µg/ml, whereas resistomycin (2), and resistoflavin (3) inhibited biofilm by more than 80% at the same concentration. The light microscopic investigation revealed that compounds 1-3 inhibit the formation of biofilms and the adhesion of bacterial cells to the surface of glass pieces. As observed by confocal laser scanning microscopy (CLSM), pigmented metabolites 1-3 disrupt the 3D architecture of biofilm-forming bacteria. Molecular docking was used to find out how compounds 1–3 bind to the active sites of the biofilm-forming proteins ClfB and CsgG in S. aureus and E. coli, respectively.