Investigating the prevalence of anti-influenza A viruses (AIV) antibodies in wild birds can provide important information for the understanding of bird exposure to AIV, as well as for prevention purposes. We investigated AIV exposure in nature by measuring the prevalence of anti-AIV antibodies in the nests and adults of an abundant and anthropophilic waterbird species common around the Mediterranean sea, the yellow-legged gull (Larus michahellis). Sampling took place in two colonies located in the gulf of Gabès in southern Tunisia: Sfax and Djerba. Antibodies were detected in the two sites, with higher prevalence in adults, eggs, and nests at Sfax than Djerba. Across both colonies, clutches that were laid later in the season, and, thus, more likely by younger parents, showed lower prevalence. Using patch occupancy modeling applied to egg clutches, we found that it is unnecessary to sample all the eggs in a given nest; nest status (antibody positive or negative) can be reliably estimated from a single egg. Differences in the density of birds, notably Larids, between the two sites may explain the observed differences in prevalence. The higher concentration of Larids in the Sfax colony could favor the transmission of AIV to yellow-legged gulls. This study highlights the importance of further developing ecological-based approaches to the factors determining the circulation of infectious agents in species such as the yellow-legged gull, which exist at the interface between diverse biological communities and human activities.