The use of Galleria mellonella as a model host to elucidate microbial pathogenesis and search for novel drugs and therapies has been well appreciated over the past years. However, the effect of microorganisms with functional appeal in the specific host remains scarce. The present study investigates the effect of treatment with selected lactic acid bacteria (LAB) with probiotic potential, as potential protective agents by using live or heat-killed cells at 6 and 24 h prior to infection with Listeria monocytogenes and Staphylococcus aureus or as potential therapeutic agents by using cell-free supernatants (CFS) after infection with the same pathogens. The employed LAB strains were Lactobacillus pentosus B281 and Lactobacillus plantarum B282 (isolated from table olive fermentations) along with Lactobacillus rhamnosus GG (inhabitant of human intestinal tract). Kaplan-Meier survival curves were plotted while the pathogen's persistence in the larval hemolymph was determined by microbiological analysis. It was observed that the time (6 or 24 h) and type (live or heat-killed cells) of challenge period with LAB prior to infection greatly affected the survival of infected larvae. The highest decrease of L. monocytogenes population in the hemolymph was observed in groups challenged for 6 h with heat-killed cells by an average of 1.8 log units compared to non challenged larvae for strains B281 (p 0.0322), B282 (p 0.0325), and LGG (p 0.0356). In the case of S. aureus infection, the population of the pathogen decreased in the hemolymph by 1 log units at 8 h post infection in the groups challenged for 6 h with heat-killed cells of strains B281 (p 0.0161) and B282 (p 0.0096) and by 1.8 log units in groups challenged with heat-killed cells of LGG strain (p 0.0175). Further use of CFS of each LAB strain did not result in any significant prolonged survival but interestingly it resulted in pronounced decrease of L. monocytogenes in the hemolymph at 24 h and 48 h after infection by more than 1 log unit (p < 0.05) depending on the strain. The results of the present work support the broader use of G. mellonella larvae as a low cost in vivo tool for screening for probiotic properties.