The skin glands of Ranidae are a rich source of antimicrobial peptides. In this study, the genomic RNA of Rana dybowskii was extracted from its skin while under Rana grylio virus stress. Five new cDNA sequences encoding 5 mature peptides, Ranatuerin-2YJ (GLMDIFKVAVNKLLAAGMNKPRCKAAHC), Dybowskin-YJb (IIPLPLGYFAKKP), Dybowskin-YJa (IIPLPLGYFAKKKKKKDPVPLDQ), Temperin-YJa (VLPLLETCSMTCWENNQTFGK), and Temperin-YJb (VLPLVGNLLNDLLGK), were obtained by reverse transcription polymerase chain reaction with a pair of degenerate primers designed according to the conserved terminal sequences of cDNA encoding antimicrobial peptide precursors of genus Rana. The antimicrobial activities of the peptides were analyzed, and the results demonstrated that all these peptides showed a significant anti-Rana grylio virus activity, and the virus was gradually cleared with the increase in gene expression. Among the 5 peptides obtained in this work, Ranatuerin-2YJ also showed a broad-spectrum anti-Gram-positive bacteria and anti-Gram-negative bacteria activity with a minimal inhibitory concentration of 22.5 µg/mL and 7.64% hemolysis activity, both of which were significantly lower (p < 0.05) than that of the other peptides. Moreover, Ranatuerin-2YJ was widely distributed in the skin, liver, spleen, and blood of R. dybowskii, while the other 4 peptides could only be cloned from the skin, indicating that the Ranatuerin-2YJ in vivo plays an important role in the protection against pathogen invasion.