Antimicrobial peptides as ancient immune system are found in almost all types of living organisms. Amphibian's skin is an important source of bioactive peptides with strong antibacterial, antiviral, and antitumor properties. They have important role in inducing apoptosis as well as cancer therapy in vitro. In this study, we extracted and purified antimicrobial peptides from skin secretions of Euphlyctis cyanophlyctis and named them brevinin-Eu and cyanophlyctin β. They showed favorable antibacterial properties on both Gram-positive and Gram-negative bacteria with ignorable hemolytic activity of <1.9% and 0.7% at very high concentrations of brevinin-Eu and cyanophlyctin β, respectively. For antibacterial activity and MIC determination, two Gram-positive (Staphylococcus aureus PTCC1431 and B. cereus PTCC1247) and two Gram-negative bacteria (Escherichia coli HP101BA 7601c and Klebsiella pneumoniae PTCC1388) were assayed. MIC values of extracted peptides demonstrated that they can inhibit bacterial growth at very low concentration (17 and 12 μg/mL) for brevinin-Eu and cyanophlyctin β, respectively. Structural prediction suggested that the brevinin-Eu can efficiently bind and destroy bacterial membrane, but cyanophlyctin β uses a diverse mode of action.