Background/Objective: Highly pathogenic avian influenza (HPAI) H5 subtype remains a significant menace to both the poultry industry and human public health. Biosecurity and mass vaccination of susceptible commercial poultry flocks are crucial to reduce the devastating economic loss and hinder the evolution of the virus. Methods: In this study, we developed a multivalent avian influenza virus (AIV) vaccine, including strains representing the HPAI 2.2.1.1., 2.2.1.2., and 2.3.4.4b clades circulating in Egypt and the Middle East. Specific pathogen-free (SPF) two-week-old chickens were vaccinated with a single vaccine shot and observed for four weeks post-vaccination before being challenged. The challenge experiment involved using one strain of HPAI H5N1 subtype clade 2.2.1.2 and two strains of HPAI H5N8 subtype clade 2.3.4.4b derived from chickens and ducks. To assess the vaccine’s potency and efficacy, the pre-challenge humoral immune response and post-challenge survival and virus shedding were evaluated. Results: All the vaccinated birds exhibited 100% seroconversion 2 weeks post-vaccination (2 WPV). In addition, protective antibody titers against each diagnostic antigen, i.e., 7.8 ± 1.8 (H5N1, clade 2.2.1.2), 10.0 ± 0.0 (H5N1, clade 2.2.1.1), and 7.5 ± 0.9 (H5N8, clade 2.3.4.4b) were detected 3 WPV. The vaccination achieved complete protection (100%) against all challenge viruses with no disease symptoms. The vaccinated birds exhibited a statistically significant reduction in oropharyngeal virus shedding 2 days post-challenge (DPC). Conclusions: This study illustrated that a single application of a multivalent genetic-matching whole AIV vaccine under laboratory conditions elicits adequate protection against the HPAI challenge, representing 2.2.1.2 and 2.3.4.4b clades. The developed vaccine has the potential to be a vaccine of choice against a broad range of HPAI in commercial flocks raised under field conditions in endemic areas.