Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (An∆pmtA, An∆pmtB, and An∆pmtC). An∆pmtA was resistant to both defensins, while An∆pmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2.