Muscotoxins are cyanobacterial cyclic lipopeptides with potential applications in biomedicine and biotechnology. In this study, CCALA125 strain extracts were enriched by polymeric resin treatment, and subjected to HPCCC affording three cyclic lipopeptides (⁻), which were further repurified by semi-preparative HPLC, affording , , and , with a purity of 86%, 92%, and 90%, respectively. The chemical identities of ⁻ were determined as muscotoxins A and B, respectively, by comparison with previously reported ESI-HRMS/MS data, whereas was determined as a novel muscotoxin variant (muscotoxin C) using NMR and ESI-HRMS/MS data. Owing to the high yield (50 mg), compound was broadly screened for its antimicrobial potential exhibiting a strong antifungal activity against , , and , with minimum inhibitory concentration (MIC) values of 0.58, 2.34, and 2.34 µg/mL; respectively, and weak antibacterial activity against with a MIC value of 37.5 µg/mL. Compounds and were tested only against the plant pathogenic fungus due to their low yield, displaying a moderate antifungal activity. The developed chromatographic method proved to be an efficient tool for obtaining muscotoxins with potent antifungal properties.