Seroin 1 and seroin 2 are abundant in silkworm cocoon silk and show strong antibacterial activities, and thus are thought to protect cocoon silk from damage by bacteria. In this study, we characterized the expression pattern of silkworm seroin 3, and found that seroin 3 is synthesized in the female ovary and secreted into egg to play its roles. After being infected, seroin 1, 2, and 3 were significantly up-regulated in the silkworm. We synthesized the full-length protein of seroin 1, 2, and 3 and their N/C-terminal domain (seroin-N/C), and compared the antimicrobial activities in vitro. All three seroins showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. Seroin 2 showed better antibacterial effect than seroin 1 and 3, whereas seroin 1/2/3-N was better than seroin 1/2/3-C. We found that seroin 2-C has stronger peptidoglycan binding ability than seroin 2-N per the ELISA test. The binding sites of seroin 2 with bacteria were blocked by peptidoglycan, which resulted in the loss of the antibacterial activity of seroin 2. Collectively, these findings suggest that seroin 1 and 2 play antibacterial roles in cocoon silk, whereas seroin 3 functions in the eggs. The three silkworm seroins have the same antibacterial mechanism, that is, binding to bacterial peptidoglycan by the C-terminal domain and inhibiting bacterial growth by the N-terminal domain.