In December 2005, the four major Swiss zoos carried out the vaccination of selected zoo birds with the adjuvant inactivated vaccine H5N2 Nobilis influenza. Pre- and post-vaccination antibody titers were determined either by hemagglutination inhibition (HI) test (non-Galliformes) or by enzyme linked immunosorbent assay (ELISA) (Galliformes) at Week 0, 5, 10, and 26 (Day 0–1, 35–36, 70–71, and 182 respectively) to determine the humoral immune response to H5 antigen. After the first vaccination, the overall geometric mean titer of non-Galliformes was 65 (n = 142), which increased to 187 (n = 139) after booster vaccination and dropped to 74 (n = 65) six months after first vaccination. For the Galliformes group, the mean titers were found to be 2.09 at Week 5 (n = 119), 3.24 at Week 10 (n = 113), and 1.20 at Week 26 (n = 39). Within the non-Galliformes, significant differences in geometric mean titers were found among different species representatives. In general, the flamingos (Phoenicopteriformes) showed a strong response to vaccination, reaching a geometric mean titer of 659 at Week 10, while the Sphenisciformes did not show high antibody titers even after booster vaccination, reaching a maximum geometric mean titer of only 65. Based on the antibody titer profiles of all investigated species, we recommend at least annual revaccination for the species that we investigated.