Background: The poultry industry has long been challenged by avian influenza which causes significant economic loss due to decreased egg production and quality. In addition, the notable ability of influenza viruses to develop resistance to conventional antibiotics is one of the biggest tasks that the industry currently faces. Attempts have been made to treat this bird flu with multiple approaches, but effective natural solutions remain elusive. Bee venom (BV) is used for the treatment of various human diseases due to its known anti-inflammatory and antibacterial properties. Recent studies suggest that antimicrobial peptides discovered in BV may be utilized as tools for the design of structurally novel antiviral agents effective against influenza viruses. Materials and Methods: In the present study, we purified BV containing 63.9% ± 5.4% melittin, 10.9% ± 1.6% phospholipase A2, and 2.3% ± 0.3% apamin. BV was evaluated in vitro for its ability to inhibit the binding of H9N2 to the chicken red blood cells. Results: We found that anti-influenza activity of BV is equivalent to that of positive control. However, we observed the neutralization of H9N2 by BV as compared to the virus only group without BV. The hypothesized anti-influenza property of BV was further examined in chicken influenza infection model. The administration of BV through intranasal route resulted in no significant antiviral effect in chickens. Conclusion: This study does not support our hypothesis that BV can reduce the viral activity in chickens.