The emergence of new avian influenza virus (AIV) strains able to infect humans represents a serious threat to global human health. In addition to surveillance and vaccine development, antiviral therapy remains crucial for AIV control; however, the increase in drug-resistant AIV strains underscores the need for novel approaches to anti-influenza chemotherapy. We have previously shown that the thiazolide anti-infective nitazoxanide (NTZ) inhibits influenza A/PuertoRico/8/1934(H1N1) virus replication, and this effect was associated with inhibition of viral hemagglutinin (HA) maturation. Herein we investigated the activity of the second-generation thiazolide haloxanide (HLN) against H5N9, H7N1 and H1N1 AIV infection in vitro, and explored the mechanism of the antiviral action. Using the A/chicken/Italy/9097/1997(H5N9) AIV as a model, we show that HLN and its precursor p-haloxanide are more effective than NTZ against AIV, with IC50 ranging from 0.03 to 0.1 μg/ml, and SI ranging from 200 to >700, depending on the multiplicity of infection. Haloxanide did not affect AIV entry into target cells and did not cause a general inhibition of viral protein expression, whereas it acted at post-translational level by inhibiting HA maturation at a stage preceding resistance to endoglycosidase-H digestion. Importantly, this effect was independent of the AIV-HA subtype and the host cell. Immunomicroscopy and receptor-binding studies confirmed that HLN-induced alterations impair AIV-HA trafficking to the host cell plasma membrane, a key step for viral morphogenesis. The results indicate that haloxanide could provide a new tool for treatment of avian influenza virus infections.