Iontophoresis (IONT) is a non-invasive technique in which a low electric current is used to enhance the penetration of charged molecules into tissue. This technique has been used in various fields of medicine, mostly in transdermal drug delivery. This study was aimed to evaluate the efficacy and the distribution profile of gentamicin using corneal IONT on infected and healthy rabbit eyes. Corneal iontophoresis of gentamicin sulfate was studied using drug-loaded disposable hydrogel probes mounted on a portable iontophoretic device, applying a low current for 60 seconds. This study confirmed that a triple iontophoretic treatment of gentamicin for only 60 seconds (0.5mA) significantly reduces the count of pseudomonas in the infected cornea to a non-infectious level. Peak gentamicin concentrations at the healthy corneas (363.1 ± 127.3 μg/g) and at the aqueous humor (29.4 ± 17.4 μg/ml) were reached immediately and two hours after a single iontophoretic treatment, respectively. The concentration versus time profile of gentamicin following iontophoresis revealed a gentamicin half life of 2.07 h in the anterior chamber, and a clearance of 1.73 μl/min from the anterior chamber to the posterior segments of the eye. This study indicates that a short iontophoretic treatment using gentamicin-loaded hydrogels has a potential clinical value in treating corneal infections, by increasing drug penetration to the eye and maintaining therapeutic levels for more than eight hours.