In Indonesia, highly pathogenic avian influenza A/H5N1 virus has become endemic and was linked to direct transmission to humans. During 2013 to 2014, we isolated avian influenza A/H5N1 and A/H3N6 viruses from poultry in Indonesia. This study aimed to reveal their pathogenicity in mammals using a mouse model. Three of the isolates, Av154 of A/H5N1 clade 2.3.2.1c, Av240 of A/H5N1 clade 2.1.3.2b, and Av39 of A/H3N6, were inoculated into BALB/c mice. To assess morbidity and mortality, we measured body weight daily and monitored survival for 20 days. Av154- and Av240-infected mice lost 25% of the starting body weight by day 7, while Av39-infected mice did not. Most of Av154-infected mice died on day 8, while majority of Av240-infected mice survived until day 20. A 50%mouse lethal dose was calculated to be 2.0x101 50%-egg infectious doses for Av154, 1.1x105 for Av240, and >3.2x106 for Av39. Av154 virus was highly virulent and lethal in mice without prior adaptation, suggesting its high pathogenic potential in mammals. Av240 virus was highly virulent but modestly lethal, and Av39 virus was neither virulent nor lethal. Several mammalian adaptive markers of amino acid residues were associated with the highly virulent and lethal phenotypes of Av154 virus.