This study evaluated the therapeutic potential of 'Pseudomonas aeruginosa' PAO1 gentamicin-induced membrane vesicles (g-MVs) against four Gram-positive organisms. These potential drug delivery devices encapsulate gentamicin and periplasmic enzymes like autolysin. Bactericidal assays and electron microscopy of thin sections revealed that 'Bacillus subtilis' 168 and 'Staphylococcus aureus' D2C were vulnerable to g-MV mediated killing, 'Listeria monocytogenes' ATCC 19113 was slightly vulnerable, whereas 'Enterococcus hirae' ATCC 9790 remained invulnerable. g-MVs were generally more potent than soluble gentamicin treatments. Negatively stained whole mounts and hydrophobic interaction chromatography revealed more MVs initially attached to hydrophilic ' B. subtilis' than to predominantly hydrophobic 'E. hirae, L. monocytogenes', and 'S. aureus'. Zymograms illustrated that all organisms except 'E. hirae' were sensitive to the 26 kDa autolysin to varying degrees. Peptidoglycan O-acetylation did not influence susceptibility to MV mediated lysis. Though not universally effective, the g-MV drug delivery system remains a promising therapeutic alternative for specific Gram-positive infections.