The Toll pathway of Drosophila melanogaster, when activated by the Beauveria bassiana fungus, directs the expression of the drosomycin and metchnikowin antimicrobial peptide genes by inducing the translocation into the nucleus of the DIF transcription factor. Accordingly, DIF mutants have been reported to have a lower resistance to fungi than control flies. However, as the longevity of non-infected DIF flies has not been measured in previous studies, it could be that survival times after infection are constrained by a low longevity. In the present study, DIF flies reared in conditions similar to those used in these previous studies had much lower survival time after infection than the control flies, but the longevity of non-infected DIF flies was also very low. Using rearing conditions controlling larval crowding, age of parents and mating status of experimental flies increased longevity of non-infected flies and survival time after infection in both strains. However, DIF flies had a similar survival time after infection as control ones or a slightly lower one, which shows that the effect of DIF is weaker than previously thought.