The gram-negative bacterium Legionella dumoffii is, beside Legionella pneumophila, an etiological agent of Legionnaires' disease, an atypical form of pneumonia. The aim of this study was to determine the antimicrobial activity of Galleria mellonella defense polypeptides against L. dumoffii. The extract of immune hemolymph, containing a mixture of defense peptides and proteins, exhibited a dose-dependent bactericidal effect on L. dumoffii. The bacterium appeared sensitive to a main component of the hemolymph extract, apolipophorin III, as well as to a defense peptide, Galleria defensin, used at the concentrations 0.4 mg/mL and 40 μg/mL, respectively. L. dumoffii cells cultured in the presence of choline were more susceptible to both defense factors analyzed. A transmission electron microscopy study of bacterial cells demonstrated that Galleria defensin and apolipophorin III induced irreversible cell wall damage and strong intracellular alterations, i.e., increased vacuolization, cytoplasm condensation and the appearance of electron-white spaces in electron micrographs. Our findings suggest that insects, such as G. mellonella, with their great diversity of antimicrobial factors, can serve as a rich source of compounds for the testing of Legionella susceptibility to defense-related peptides and proteins.