In this study, we evaluated the activity of short antimicrobial peptides against different fungal isolates that cause postharvest decay of fresh fruits. The previously identified hexapeptides PAF19, PAF26 and LfcinB4-9 inhibited the in vitro growth of isolates from Penicillium digitatum and P. italicum, and from Alternaria and Geotrichum genera, being no active against Rhizopus, Mucor and Aspergillus. The results extend our previous observations on the specific and distinct activity profiles of this class of antifungal peptides. In addition, peptide activities were compared with that of two fungicides used for citrus fruit preservation, thiabendazole (TBZ) and imazalil (IMZ). We observed a lack of correlation between peptide and fungicide sensitivity among different species. Importantly, P. digitatum and P. italicum isolates resistant to fungicides were susceptible to peptides and our data suggest that common multiple drug resistance mechanisms are not active against this class of peptides. The in vitro peptide inhibition was correlated with a retard of the decay caused by Penicillium on citrus fruits, and this effect was comparable for both fungicide-resistant and -sensitive isolates. Comparison of PAF26 and TBZ in vitro minimum inhibitory concentration (MIC) values and their in vivo effect on citrus decay indicated that PAF26 performed in vivo better than TBZ.