PURPOSE: Although there have been reports describing the use of 1% voriconazole eye drops in the treatment of fungal infections, little is known about the penetration of voriconazole eye drops into the vitreous humour. The aim of this study was to elucidate if topical application of 1% voriconazole eye drops could reach therapeutic levels in the vitreous humour. METHODS: A prospective, open-label trial involving 10 participants selected from patients scheduled to undergo elective, posterior segment surgery. Participants received 1% voriconazole solution, preserved with 0.01% benzalkonium chloride, hourly for four doses or four times a day for 3 days. Vitreous humour was obtained at the start of surgery and analysed using a validated high-performance liquid chromatography assay. RESULTS: The minimum and maximum voriconazole concentration after hourly dosing (n = 5) was 0.1 and 1.1 microg/mL, respectively, whereas the median was 0.3 microg/mL. Samples were taken between 0.8 and 5.4 h after the last dose, with the median at 1.2 h. Almost all voriconazole concentrations from the four times a day group (n = 5) were below the limit of quantification. CONCLUSIONS: One per cent voriconazole eye drops are able to penetrate into human vitreous humour. Adequate concentration for treatment of sensitive Candida species can be achieved upon hourly administration.