Drosophila employs various antimicrobial peptides as effective weapons to defend against diverse pathogens. Drosomycin is an inducible antifungal peptide initially isolated from the Drosophila melanogaster haemolymph. Here we report the expression pattern of seven drosomycin genes in four different developmental stages (egg, larva, pupa and adult). Results show that drosomycin and drosomycin-2 are expressed in larva, pupa and adult, whereas drosomycin-1 and drosomycin-6 were not detected in all the stages. Moreover, all the seven drosomycin genes are shut off in egg. Functional comparison of recombinant drosomycin and drosomycin-2, both with identical expression pattern, produced from Escherichia coli, revealed their significant differences in potency against a specific fungal species. In addition, we found for the first time that drosomycin and drosomycin-2 both are antiparasitic peptides which show inhibitory effect on the ookinete development of the parasite Plasmodium berghei with differential potency. Functional differentiation between them was further evaluated by evolutionary trace analysis which identified two evolutionary epitopes (named alpha- and gamma-patch, respectively) and an important site in the m-loop. Substitutions in these regions are possibly associated with the antifungal and antiparasitic potency difference among members of the drosomycin family.