Abstract: In order to standardize the evaluation methods of antimicrobial peptides against filamentous fungi,polymyxin B was selected as the antifungal peptide,Aspergillus niger ATCC 16404 and Pencillinm chrysogenum ATCC 10106 were chosen as the bioindicators of fungi to determine the measure method in this study. The evaluation methods of polymyxin B against filamentous fungi were determined on czapek’s medium(20 mL medium per 90 mm petri dish)by comparing with two common methods of conidial germination inhibition and mycelial growth inhibition. The suitable determination conditions were then further optimized. The results showed that the evaluation method of mycelial growth inhibition was more suitable for the quantitative determination of polymyxin B against filamentous fungi. The optimal test conditions for polymyxin B against A. niger ATCC 16404 were as follows:The culture time of fungal pathogen was 48 h,the diameter of mycelium plug was(8.00±0.02)mm,and the EC₅₀ of polymyxin B was 0.68 mg/mL under these conditions. The suitable conditions for polymyxin B against P. chrysogenum ATCC 10106 were as follows:The culture time was 72 h and the mycelium plug diameter was(5.80±0.02)mm,and the EC₅₀ of polymyxin B was 0.45 mg/mL. The determination method of antifungal activity for antimicrobial peptides against filamentous fungi had a widely applicability,and it could provide a new foundation for the standard and establishment of determination method on the antimicrobial peptide against filamentous fungi.