Gentamicin is used in an ointment form for the treatment of skin infections. To investigate the effect of gentamicin used as an ointment, the antimicrobial susceptibilities against Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, and Pseudomonas aeruginosa isolated from community and medical settings were studied and compared with other antibacterial agents such as fradiomycin, chloramphenicol, and bacitracin used as active ingredient for each ointment. Gentamicin showed antibacterial activities for all standard bacteria tested, but fradiomycin and chloramphenicol showed no such activities for St. pyogenes and P. aeruginosa, respectively. Bacitracin showed activity for St. pyogenes only. The strains of staphylococci isolated from healthy people were highly susceptible to gentamicin, while 49.3% of the isolates from the patients with skin infections were resistant to gentamicin and 96.4% of the gentamicin-resistant staphylococci carried the aminoglycoside-resistance gene aacA-aphD. The growths of all strains tested, except for two strains of P. aeruginosa, were inhibited by close below 128 µg/ml of gentamicin. Furthermore, the frequencies of spontaneous mutants resistant to gentamicin, fradiomycin, and chloramphenicol were each investigated using S. aureus, S. epidermidis, St. pyogenes, and P. aeruginosa. At doses of more than 32 µg/ml of gentamicin, no resistant mutants in any of bacteria strains tested were obtained. The concentration of gentamicin on the skin was calculated at approximately 895 µg/ml at least when the commercially used 0.1% gentamicin ointment was applied to the skin. Therefore, our study strongly indicates that the gentamicin ointment used has a potency of sufficiently inhibiting the growth of bacteria, including gentamicin-resistant strains, which cause skin infections in the community.