The recent global challenges to prevent and treat fungal infections strongly demand for the development of new antifungal strategies. The structurally very similar cysteine-rich antifungal proteins from ascomycetes provide a feasible basis for designing new antifungal molecules. The main structural elements responsible for folding, stability and antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and antifungal active protein. NFAP and its mutants carrying cysteine deletion (NFAPΔC), hydrophobic core deletion (NFAPΔh), and N-terminal amino acids exchanges (NFAPΔN) were produced in Pichia pastoris. The recombinant NFAP showed the same features in structure, folding, stability and activity as the native protein. The data acquired with mass spectrometry, structural analyses and antifungal activity assays of NFAP and its mutants proved the importance of the disulphide bonding, the hydrophobic core and the correct N-terminus for folding, stability and full antifungal function. Our findings provide further support to the comprehensive understanding of the structure-function relationship in members of this protein group.