Adenoviral ocular infections are the most common external ocular infections world wide and there is no approved treatment. Topical cidofovir has been shown to be effective in vitro, in animal models and in case studies for the treatment of adenoviral ocular infections. Prophylaxis to prevent transmission within households and to reduce community epidemics remains an important public health goal. The current study examined whether antiviral prophylaxis with cidofovir, twice daily dosing, would restrict viral replication following a large challenge inoculum of adenovirus type 5 (Ad5) in the New Zealand white rabbit ocular model. The results showed that antiviral prophylaxis with 1 and 0.5% cidofovir significantly reduced mean daily Ad5 ocular titers (days 0-5), the number of Ad5 positive cultures/total (days 1-14), serial Ad5 positive cultures/total (days 1, 2, 3, 4, 5, 7), and the number of eyes with Ad5 replication beyond day 0 (1% cidofovir only). Antiviral prophylaxis appears to be an effective strategy to reduce and restrict adenovirus replication experimentally.