H6N6 avian influenza virus (AIV) has emerged as a predominant subtype in recent years, with frequent isolations reported in China, Vietnam, and South Korea. As critical reservoirs, waterfowl play a pivotal role in the maintenance and transmission of H6N6 AIV. In this study, serological surveillance and virus isolation were conducted on affected breeding duck farms and at live bird markets, resulting in the successful isolation of a novel H6N6 strain. Phylogenetic analysis revealed that this isolate is a triple-reassortant virus, with its genome originating from three distinct genetic clades. Notably, key amino acid substitutions A138S and A222V were identified in the hemagglutinin (HA) protein. In vivo challenge experiments demonstrated that the virus could infect multiple species, including mice, specific-pathogen-free (SPF) chickens, and young Muscovy ducks, inducing mild clinical lesions. Crucially, infection in breeding ducks led to significant reproductive tract damage and a decline in egg production. These findings underscore the potential of low-pathogenic H6N6 AIV to cross species barriers and impair the productivity of breeding flocks, highlighting the urgent need for sustained epidemiological surveillance.