Keratitis is a severe condition characterized by inflammation of the cornea following a local trauma. The most common ocular disease is the bacterial one, which requires an antibiotic treatment. The major limitation of this therapy is the resistance of the antibiotic. For this reason, alternative procedures have been developed and consist of antimicrobial molecules. One of the most used is the chlorhexidine gluconate, which has shown activity versus Gram-positive and Gram-negative bacteria and fungi. In addition to its efficiency, chlorhexidine shows low toxicity levels for mammalian cells and is a low-cost molecule. Despite its multiple benefits, chlorhexidine, if used at concentrations higher than 0.02% (w/w), can cause local eye irritation. Additionally, its poor penetrability through the cornea makes necessary frequent instillation of eye drops for a prolonged time. Due to these limitations, alternative drug delivery strategies are required. Here, we report a novel formulation based on the combination of d-alpha-tocopherol polyethylene glycol 1000 succinate with chlorhexidine, which results in higher accumulation of the drug in human corneas measured by liquid chromatography and strong antimicrobial activity. Moreover, this formulation does not cause any toxic effect on human cells and is well tolerated by rabbit eyes. Therefore this novel formulation represents a good candidate for the treatment of keratitis that overcomes the risk of antibiotic resistance.