A serological survey was performed in the Guadalquivir Marshes (Southern Spain) in order to determine the presence and diffusion of six infective agents in wild waterfowl. The analysis covered 712 waterfowl from 13 species belonging to five taxa (Podicipediformes, Ciconiiformes, Anseriformes, Gruiformes, and Charadriiformes). A range of immunological techniques led to detection of antibodies against the six infective agents studied: Chlamydia psittaci (13.3%), Mycoplasma anatis (3.5%), Pasteurella anatipestifer (2.9%), duck plague virus (1.6%), Aspergillus fumigatus (1.1%), and Newcastle disease virus (NDV) (0.2%). The survey results indicated that these infective agents exist and circulate among wild waterfowl in the Guadalquivir Marshes.