Topical administration of gentamicin, a hydrophilic aminoglycoside antibiotic, is limited by membrane impermeability and toxicity concerns. The purpose of this study was to develop and evaluate the antimicrobial activities of an alternative non-invasive, convenient and cost-effective transdermal drug delivery system (TDDS) containing gentamicin in biodegradable polyester-based matrices. The patches were formulated by solvent evaporation technique using PURASORB ® polymers and evaluated for thermal properties, drug content, physicochemical performance, stability, skin irritation on rat skin and antimicrobial activities against five micro-organisms: Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The differential scanning calorimetry (DSC) results indicated compatibilty between the drug and the polymers. In addition, the formulations showed good drug encapsulation, stability, physicochemical properties, tolerability on rabbit skin and higher zones of inhibition compared with a commercially available gentamicin sulphate cream against S. aureus, E. coli, S. typhi and P. aeruginosa, while K. pneumoniae was mildly susceptible. Compared with the rest of the formulations, patches of PURASORB ® PL 32 exhibited the best stability, tolerability on rat skin and bioactivity. This study has shown that transdermal patches of PURASORB ® PL 32 represent an alternative delivery system for gentamicin for treatment of infections caused by gentamicin-susceptible micro-organisms.