Patagonia in southern South America is among the few world regions where direct human impact is still limited but progressively increasing, mainly represented by tourism, farming, fishing and mining activities. The sanitary condition of Patagonian wildlife is unknown, in spite of being critical for the assessment of anthropogenic effects there. The aim of this study was the characterization of Salmonella enterica strains isolated from wild colonies of Magellanic penguins (Spheniscus magellanicus) located in Magdalena Island and Otway Sound, in Chilean Patagonia. Eight isolates of Salmonella were found, belonging to Agona and Enteritidis serotypes, with an infection rate of 0·38%. Resistance to ampicillin, cefotaxime, ceftiofur and tetracycline antimicrobials were detected, and some of these strains showed genotypic similarity with Salmonella strains isolated from humans and gulls, suggesting inter-species transmission cycles and strengthening the role of penguins as sanitary sentinels in the Patagonian ecosystem.