Sardinia is a Mediterranean island with a long geological history, leading to a separation process from continental Europe during the Miocene. As a consequence, in this insular habitat some wild bird species developed endemic forms, some of which are currently threatened. The aim of this study is to evaluate the possible animal health risk associated with a potential avian influenza virus (AIV) circulation in Sardinian wild bird populations. Overall, 147 cloacal swabs were sampled in the Sardinia region from June 2009 to September 2011. Samples were obtained from 12 taxonomic orders, including 16 families and 40 species of birds. Based on the endangered host status or on the ecology of the host-virus interaction, samples were categorized into three groups of species: 1) endemic, endangered, or both (17 species); 2) potential reservoir (21 species); and 3) potential spillover (two species). Cloacal swabs were tested by reverse transcription (RT)-PCR for influenza A virus matrix gene amplification. Forty-one serum samples were tested by nucleoprotein-enzyme-linked immunosorbent assay (NP-ELISA) for antibodies against influenza A virus nucleoprotein and by hemagglutination inhibition assay for detection of seropositivity against H5 and H7 AIV subtypes. No cloacal swabs tested RT-PCR positive for AIV, whereas two weak seropositive results were detected by NP-ELISA in a mallard (Anas platyrhynchos) and in a yellow-legged gull (Larus michahellis). The low or absent AIV circulation detected in Sardinia's wild birds during the study suggests a naїve status in these avian populations. These data provide new information on AIV circulation in Sardinia's wild birds that could be applied to implement conservation strategies for threatened species.