The number of multidrug-resistant pathogenic microorganisms has been growing in recent years, most of which is due to the inappropriate use of the commercial antibiotics that are currently available. The dissemination of antimicrobial resistance represents a serious global public health problem. Thus, it is necessary to search for and develop new drugs that can act as antimicrobial agents. Antimicrobial peptides are a promising alternative for the development of new therapeutic drugs. Anurans’ skin glands are a rich source of broad-spectrum antimicrobial compounds and hylids, a large and diverse family of tree frogs, are known as an important source of antimicrobial peptides. In the present study, two novel antimicrobial peptides, named Raniseptins-3 and -6, were isolated from Boana raniceps skin secretion and their structural and biological properties were evaluated. Raniseptins-3 and -6 are cationic, rich in hydrophobic residues, and adopt an α-helix conformation in the presence of SDS (35 mM). Both peptides are active against Gram-negative bacteria and Gram-positive pathogens, with low hemolytic activity at therapeutic concentrations. No activity was observed for yeasts, but the peptides are highly cytotoxic against B16F10 murine melanoma cells and NIH3T3 mouse fibroblast cells. None of the tested compounds showed improvement trends in the MTT and LDH parameters of MHV-3 infected cells at the concentrations tested.