Chlorhexidine digluconate (CHG) is a widely used antiseptic for skin disinfection, as it exhibits poor skin permeability. Therefore, aim of the present study was preparation and evaluation of CHG nanoemulsions (NEs) using high-speed homogenisation (HSH) combined with ultra-probe sonication to improve skin permeability. CHG-NEs were prepared using eucalyptus oil (EO) and olive oil (OO), with a surfactant-to-cosurfactant ratio of 2:1. Optimised NEs had a mean droplet diameter of 257.5 ± 12.4 nm and PDI of 0.56 ± 0.13. In In vitro permeation studies EO based NEs delivered more CHG into the skin, 6.15 ± 0.12 µg/mg compared to 3.01 ± 0.02 µg/mg for a control solution. Additionally NEs were incorporated into an in situ forming dressing and results showed controlled delivery of CHG within skin. Together, these findings bring new possibilities for topical antimicrobial treatment and prophylactic strategies in wound management.