Biofilm-forming bacteria have sent alarms to the world about the emerging of bacterial resistance. Gentamycin is an aminoglycoside broad-spectrum antibiotic used against microbial infections. The transdermal drug delivery method is a major system used to reduce drug toxicity and avoid first-pass metabolism. Gentamycin was formulated in a transdermal polymeric formula using hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone (PVP), and Chitosan in the presence of palmitic acid as a permeation enhancer. In this research, gentamycin extended drug release behavior was successfully done in different polymeric formulas containing (HPMC/PVP) and (HPMC/Chitosan), with a maximum drug release of <70%. In addition, drug diffusion was found to be dependent on the rate of drug release. The controlled release formulas selected for antimicrobial assay show that HPMC/Chitosan formulas have successfully inhibited microbial and biofilm growth by 90%. In conclusion, gentamycin can be formulated in a transdermal polymeric film to target skin infection, reduce drug side effects and avoid drug first-pass metabolism.