AbstractAs a consequence of the fast resistance spreading, a limited number of drugs are available to treat fungal infections. Therefore, there is an urgent need to develop new antifungal treatment strategies. The features of a disulfide bond‐stabilized antifungal protein, NFAP2 secreted by the moldNeosartorya(Aspergillus)fischerirender it to be a promising template for future protein‐based antifungal drug design, which requires knowledge about the native disulfide linkage pattern as it is one of the prerequisites for biological activity. However, in the lack of tryptic and chymotryptic proteolytic sites in the ACNCPNNCK sequence, the determination of the disulfide linkage pattern of NFAP2 is not easy with traditional mass spectrometry‐based methods. According toin silicopredictions working with a preliminary nuclear magnetic resonance (NMR) solution structure, two disulfide isomers of NFAP2 (abbaccandabbcac) were possible. Both were chemically synthesized; and comparative reversed‐phase high‐performance liquid chromatography, electronic circular dichroism and NMR spectroscopy analyses, and antifungal susceptibility and efficacy tests indicated that theabbcacis the native pattern. This knowledge allowed rational modification of NAFP2 to improve the antifungal efficacy and spectrum through the modulation of the evolutionarily conserved γ‐core region, which is responsible for the activity of several antimicrobial peptides. Disruption of the steric structure of NFAP2 upon γ‐core modification led to the conclusions that this motif may affect the formation of the biologically active three‐dimensional structure, and that the γ‐core modulation is not an efficient tool to improve the antifungal efficacy or to change the antifungal spectrum of NFAP2.