Nocardiosis is an emerging zoonosis caused by Nocardia spp. bacteria. The disease is linked to infections in immunocompromised individuals, and is potentially lethal when it turns systemic and is left untreated. The development of reliable and valid diagnostic methods is crucial to the identification of these pathogenic agents, particularly in wild mammals, which are potential reservoirs. The aim of this study was to determine the molecular prevalence of Nocardia spp. in wild mammals in Portugal. A sample of 114 wild mammals belonging to the orders Canidae, Cervidae, Erinaceidae, Herpestidae, Leporidae, Mustelidae, Viverridae, and Suidae were studied. Tissue samples (n=206) were collected from different organs. The molecular prevalence of Nocardia in wild mammals was 55.3% (95% CI: 45.7-64.6%). Nocardia spp. were detected in nine of ten species studied: 83.3% (95% CI: 62.2-100%) in Egyptian mongoose (Herpestes ichneumon), 63.2% (95% CI: 41.5-89.9%) in red fox (Vulpes vulpes), 45.9% (95% CI: 29.8-62.0%) in red deer (Cervus elaphus), 44.4% (95% CI: 28.2-60.6%) in wild boar (Sus scrofa), 3/3 beech martens (Martes foina), 2/2 Eurasian otters (Lutra lutra), 1/1 European badger (Meles meles), 1/1 genet (Genetta genetta), and 1/2 hedgehog (Erinaceus europeaeus). Of the 206 tissue samples studied by molecular techniques, Nocardia spp. were detected in 37.4% (95% CI: 36.7-38.1%), with a predominance in mesenteric lymph nodes (13.1%; 95% CI: 12.4-13.9%) and kidneys (9.2%; 95% CI: 8.5-9.9%). These results provide new insight into the prevalence of Nocardia in wild mammals and highlight the need for surveillance of wildlife as a potential reservoir of these emergent pathogens.