Purpose: Adenoviral conjunctivitis is the most common cause of conjunctivitis 
worldwide with no approved antiviral treatment. Benzalkonium chloride (BAK) is a 
common preservative in ophthalmic medications and is the active ingredient in 
some skin disinfectants and hand sanitizers. BAK is known to be effective in 
killing bacteria and enveloped viruses; however, its activity against ocular 
types of nonenveloped adenoviruses (Ads) is unknown. The goal was to determine 
whether BAK is an effective antiviral agent against common human ocular types of 
adenovirus in vitro. Methods: The direct inactivating activity of BAK was 
determined by incubating several human adenovirus types with BAK concentrations 
of 0.001%, 0.003%, 0.005%, 0.01%, 0.1%, and 0% for 1 h at 33°C. Resulting 
adenovirus titers were determined after treatment. Decreases in titers of ≥3 
Log10 were considered virucidal, while decreases in titers of <1 Log10 were 
considered ineffective. Results: BAK 0.1% was virucidal for Ad3, Ad5, Ad7a, 
Ad19/64, and Ad37, while it reduced titers >1 Log10, but <3 Log10 for Ad4 and 
Ad8. Decreases in titers >1 Log10 were demonstrated for BAK 0.003%, 0.005%, and 
0.01% for Ad5 only. Decreases in titers for the other adenovirus types for those 
concentrations were ≤0.53 Log10. 0.001% BAK produced minimal decreases in titers 
for all types. Conclusions: BAK, at 0.01% or less was not consistently effective 
as an antiviral against adenovirus, but higher concentrations, such as 0.1%, 
should be further investigated as a possible topical treatment for adenoviral 
ocular infections, providing ocular toxicity is not an issue.