Proteins and peptides belonging to the plant immune system can possess natural antibacterial, antifungal and antiviral properties. Due to their broad range of activity and stability, they represent promising novel alternatives to commonly used antifungal agents to fight the emergence of resistant strains. An isolation protocol was optimised to target proteins found in plants' defence system, and it was applied to white mustard (Brassica hirta) seeds. Firstly, a ∼14 kDa protein with activity against S. cerevisiae was extracted and purified; secondly, the protein was identified as the mustard Napin protein named Allergen Sin a 1. Napin is the name given to seed storage (2S) albumin proteins belonging to the Brassicaceae family. While several Napins have been described for their antimicrobial potential, Sin a 1 has been mainly studied for its allergenic properties. The antimicrobial activity of Sin a 1 is described and characterised for the first time in this study; it possesses antifungal and antiyeast in vitro activity, but no antibacterial activity was recorded. The yeasts Zygosaccharomyces bailii Sa 1403 and Saccharomyces cerevisiae DSM 70449 along with the filamentous fungi Fusarium culmorum FST 4.05 were amongst the most senstitive strains to Sin a 1 (MICs range 3-6 μM). The antimicrobial mechanism of membrane permeabilisation was detected, and in general, the antifungal activity of Sin a 1 seemed to be expressed in a dose-dependent manner. Data collected confirmed Sin a 1 to be a stable and compact protein, as it displayed resistance to α-chymotrypsin digestion, heat denaturation and insensitivity to pH variations and the presence of salts. In addition, the protein did not show cytotoxicity towards mammalian cells.