Purpose <p><i>Trichophyton mentagrophytes</i> is a common causative factor of dermatophytosis in humans and animals, and its increasing drug resistance challenge has attracted widespread attention. Given their inherent low drug resistance, antimicrobial peptides offer a promising method for addressing drug resistance of <i>T. mentagrophytes</i>. ML-AMP1 is a peptide with high antifungal activity against <i>T. mentagrophytes</i>. This study aimed to evaluate its antifungal activity against <i>T. mentagrophytes</i> and to determine its potential as a drug application.</p> Methods <p>ML-AMP1 was synthesized via solid-phase peptide synthesis, and its antifungal activity was evaluated through minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and time-kill kinetics assays. The safety and resistance induction of ML-AMP1 were tested by cytotoxicity and resistance induction assays. The mechanism of action of ML-AMP1 was explored using light microscopy, fluorescence staining and TEM. An animal dermatophyte model was constructed to evaluate the in vivo efficacy of ML-AMP1.</p> Results <p>The MIC and MFC of ML-AMP1 against <i>T. mentagrophytes</i> were 62.31&#xa0;µg/mL and 125.00&#xa0;µg/mL (2×MIC), respectively. At 2×MIC, ML-AMP1 eradicated <i>T. mentagrophytes</i> within 12&#xa0;h. Its IC₅₀ against mouse fibroblasts (L929) was 8.14 times higher than its MIC. Resistance induction assays showed no resistance development in <i>T. mentagrophytes</i>. ML-AMP1 inhibited microconidia germination, disrupted fungal cell membrane integrity, and induced fungal death through ROS overproduction. In vivo, ML-AMP1 restored infected skin to health.</p> Conclusion <p>The results indicate that ML-AMP1 has the potential to be developed into a novel antifungal drug for dermatophytosis.</p> Graphical Abstract <p></p>