Avian influenza virus (AIV) H9N2 was declared to be endemic in birds of the Middle East, in particular in Egypt, with multiple cases of human infections. Despite concerns about the pandemic threat posed by H9N2 AIV, due to the fact that its receptor specificity is similar to that of human influenza viruses, its morbidity and mortality rates in humans are so far negligible. However, the acquisition of specific adaptive amino acid (aa) mutations in the viral polymerase can enhance cross-species transmission of the virus itself or of reassortants, which gained these changes. The polymerase basic protein 2 (PB2) is one of the key determinants for AIV adaptation towards mammals. Although mammalian pathogenicity-related mutations (MPMs) in PB2 genes were identified in different AIVs, the specific effect of single or multiple mutations on viral fitness has not been compared so far. Here, we studied the effect of the aa K at position 591, which was frequently reported in the PB2 of Egyptian H9N2 isolates, on the proliferation efficiency and polymerase activity of an H5N1 (clade 2.2.1.2) AIV already carrying the mammalian adaptive mutation 627K. Using reverse genetics, we generated a set of recombinant parental strains and H5N1 variants carrying the avian-like 591Q/627E or mammalian-like adaptive mutations 591K/627K (H5N1EGY, H9N2EGY, H5N1PB2-H9N2EGY, H5N1H9N2_PB2_K591Q, H5N1PB2_K627E, H5N1PB2_K627E/591K, H5N1PB2_627K/591K). Regardless of the avian-like 627E or the mammalian-adaptive 627K, both variants carrying the 591K (H5N1PB2_K627E/591K, H5N1PB2_627K/591K) and the reassortant H5N1PB2-H9N2EGY replicated to significantly higher levels in mammalian continuous MDCK and Calu-3 cell lines and primary normal human bronchial epithelial cells than the parental H5N1EGY virus (carrying solely the 627K adaptive mutation). Expectedly, the H5N1 variants carrying avian-like PB2 mutations (H5N1H9N2_PB2_K591Q, H5N1PB2_K627E) replicated to significantly lower levels than the parental H5N1EGY virus in the predefined primary and continuous mammalian cell line systems. Consistently, the activity of H5N1 subtype AIV polymerase complexes comprising PB2 segments with singular 591K or combined with 627K was significantly enhanced when compared to parental H5N1EGY and H9N2EGY. This study emphasizes the significant impact of 591K containing PB2 segments in the background of H5N1 polymerase on viral fitness in addition to the well-known MPM 627K in vitro.