Most clinically isolated strains are drug-resistant, emphasizing the urgent need to discover alternative therapies. In this study, the previously characterized Octominin was modified into a shorter peptide with an 18 amino acid sequence (GWLIRGAIHAGKAIHGLI) and named Octominin II. The secondary structure of Octominin II is a random coil with a helical turn and a positive charge (+2.46) with a hydrophobic ratio of 0.46. Octominin II inhibited , , and with minimum inhibitory and fungicidal concentrations against of 80 and 120 µg/mL, respectively. Field emission scanning electron microscopy confirmed that Octominin II treatment caused ultra-structural changes in cells. Furthermore, membrane permeability results for the fluorescent indicator propidium iodide revealed modifications in cell wall integrity in Octominin II-treated . Octominin II treatment increases the production of reactive oxygen species (ROS) in . Gene expression studies revealed that Octominin II suppresses virulence genes of such as , , , , , and . In addition, a nucleic acid binding assay revealed that Octominin II degraded genomic DNA and total RNA in a concentration-dependent manner. Additionally, Octominin II inhibited and eradicated biofilm formation. Octominin II showed relatively less cytotoxicity on raw 264.7 cells (0-200 µg/mL) and hemolysis activity on murine erythrocytes (6.25-100 µg/mL). In vivo studies confirmed that Octominin II reduced the pathogenicity of . Overall, the data suggests that Octominin II inhibits by employing different modes of action and can be a promising candidate for controlling multidrug-resistant infections.