Though influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subject to aerosol inoculation. The temperature at which cells were cultured post-inoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to 37°C and this effect was slightly greater for the conjunctivitis isolates than the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multi-layer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited hemagglutination of both ocular and non-ocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of non-ocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells, but that this is more likely for ocular tropic viruses as they are better able to maintain their infectivity during passage through the tear film.IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza viral tropism have yet to be fully elucidated. Here, we sought to identify factors which limit the ability of most influenza viruses to cause ocular infection. Though ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors which influence infection and replication in this non-canonical site may point toward novel determinants of tropism in the respiratory tract.