Highly pathogenic (HP) and low pathogenic (LP) avian influenza viruses (AIVs) belonging to H5 and H7 subtypes have been found to be associated with human infection as the result of direct transmission from infected poultry. Human infections by AIVs can cause mild or subclinical disease, and serosurveys are believed to represent an important tool to identify risk of zoonotic transmission. Therefore, we sought to examine Italian poultry workers exposed during LPAI and HPAI outbreaks with the aim of assessing serologic evidence of infection with H5 and H7 AIVs. From December 2008 to June 2010 serum samples were collected from 188 poultry workers and 379 nonexposed controls in Northern Italy. The hemagglutination inhibition (HI) assay using horse red blood cells (RBCs) and a microneutralization (MN)–enzyme-linked immunosorbent assay test were used to analyze human sera for antibodies against the following H5 and H7 LPAI viruses: A/Dk/It/4445/07(H5N2); A/Ty/It/2369/09(H5N7); A/Ty/It/218-193/10; A/Ck/It/3775/99(H7N1); A/Ty/It/214845/03(H7N3); and A/Dk/It/332145/09(H7N3). Since previous studies identified low antibody titer to AIVs in people exposed to infected poultry, a cutoff titer of ≥1∶10 was chosen for both serologic assays. Only HI-positive results confirmed by MN assay were considered positive for presence of specific antibodies. The Fisher exact test was used to analyze differences in seroprevalence between poultry workers and control groups, with the significance level set at P < 0.05. MN results showed a proportion of H7-seropositive poultry workers (6/188, i.e., 3.2%), significantly higher than that of controls (0/379), whereas no MN-positive result was obtained against three H5 LPAI subtypes recently identified in Italy. In conclusion, the survey indicated that assessing seroprevalence can be an important tool in risk assessment and health surveillance of poultry workers.