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Simple Summary A retrospective study was carried out on the presence of Flavivirus antibodies in the European wild boar (Sus scorfa) in the Lazio region of Italy. 168 serum samples from European wild boars were tested by cELISA, virus neutralization test (VNT), and RT-PCR. All sera that had cELISA anti-Flavivirus antibodies were further examined by the VNT. Thirteen wild boars (7.73%) were positive for Flavivirus by cELISA, a single European wild boar serum was positive for VNT, with a neutralizing antibody titer 1/14. No samples resulted positive for Flavivirus by RT-PCR assay. Abstract Background: A retrospective sero-survey for evidence of West Nile virus (WNV) infection in European wild boar (Sus scorfa) was conducted in the Latium region, Italy, on stored serum samples of the period November 2011 to January 2012. Methods: Sera were collected from 168 European wild boars and screened for antibodies to WNV and other Flaviviruses by competitive enzyme linked immunosorbent assay (cELISA). All sera positive for Flavivirus antibodies by cELISA were further examined by virus neutralization test (VNT). To test the presence of Flavivirus RNA in samples, an RT-PCR was performed using a pan-Flavivirus primers pair. Results: Thirteen wild boars (7.73%) were seropositive for Flaviviruses. The hemolysis of serum samples limited the interpretation of the VNT for 7 samples, confirming the presence of specific antibody against WNV in a single European wild boar serum sample. The presence of ELISA positive/VNT negative samples suggests the occurrence of non-neutralizing antibodies against WNV or other antigen-related Flaviviruses. No samples resulted positive for Flavivirus by RT-PCR assay. Conclusion: Although a moderately high percentage of animals with specific antibody for WNV has been detected in wild boar in other surveillance studies in Europe, this has not been reported previously in Italy. Together, these data indicate that European wild boar are exposed to WNV and/or other related-Flavivirus in central Italy and confirm the usefulness of wild ungulates, as suitable Flavivirus sentinels.