The Bee venom peptide Anoplin (GLLKRIKTLL) was synthesized and modified by using antibiotics at its N-terminus, resulting in three peptide derivatives: Ano1, Ano2 and Ano3. The synthetic yields were 92.3%, 75.1% and 95.4%, respectively. Multi-spectroscopy methods were employed to investigate the interaction between these peptides and ct-DNA. The experimental results revealed that Anoplin, Ano1 and Ano2 interacted with ct-DNA in a groove-binding mode, whereas Ano3 exhibited a mosaic-binding mode. Moreover, circular dichroism revealed that these peptides have ability to unfold parallel G-quadruplex structures, indicating that they can interact with secondary nucleic acid structure. Notably, antimicrobial activity results indicated that all three derived peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. The synthesized peptide conjugate Ano3 exhibited a MIC value of 1.4 μM to S. flexneri. Scanning electron microscopy results distinctly showed that Ano3 could rupture the cell wall of bacteria. These results provide novel methods to create effective antibacterial agents for both Gram-positive and Gram-negative bacteria by utilizing natural toxic molecules.