The antibacterial activity of hemolymph from Galleria mellonella infected with entomopathogenic strain of Pseudomonas aeruginosa and non-pathogenic bacterium Escherichia coli was studied. In vivo, the antimicrobial activity appeared shortly after P. aeruginosa infection, reached the maximum level 18 h postinjection, while 30 h later only trace activity was noted. The activity induced by E. coli sustained on the high level until 48 h after infection. We also noted that the antimicrobial activity level induced by the non-pathogenic bacterium was higher in comparison to that measured in insects infected with the pathogenic strain of P. aeruginosa. The results of our in vitro studies indicated that inducible antimicrobial peptides of G. mellonella larvae were digested by P. aeruginosa elastase B. After 1 h incubation of cell-free hemolymph of immune-challenged larvae with elastase B, no antibacterial activity was observed. It was also shown that elastase B degraded synthetic cecropin B while in the presence of 6 mM EDTA antibacterial activity of cell-free hemolymph as well as cecropin B, was not changed which confirmed that the activity was abolished by the metalloprotease.