Summary Previously, it was shown that the Arabidopsis thaliana plant defensins AtPDF1.1 (At1g75830) and AtPDF1.2a (At5g44420) exert in vitro antimicrobial properties and that their corresponding genes are expressed in seeds and induced in leaves upon pathogen attack, respectively. In this study, the expression profile of both AtPDF1.1 and AtPDF1.2a is analysed in wild‐type plants upon different stress‐related treatments and the effect of modulation of their expression in transgenic plants is examined in both host and nonhost resistance. AtPDF1.1, which was originally considered to be seed‐specific, is demonstrated to be locally induced in leaves upon fungal attack and exhibits an expression profile distinct from that of AtPDF1.2a, a gene frequently used as marker for the ethylene/jasmonate‐mediated signaling pathway. Transgenic plants with modulated AtPDF1.1 or AtPDF1.2a gene expression show no altered phenotype upon Botrytis cinerea inoculation. However, constitutive overexpression of AtPDF1.1 in A. thaliana leads to a reduction in symptoms caused by the nonhost Cercospora beticola causing non‐spreading spots on A. thaliana leaves. These results indicate that AtPDF1.1 and AtPDF1.2a clearly differ regarding their expression profile and functionality in planta. It emphasizes the additional level of complexity and fine‐tuning within the highly redundant plant defensin genes in A. thaliana.