Anthropogenic activity in Antarctica constitutes a continuous risk for the introduction of infectious diseases into indigenous wildlife populations. Penguin colonies living close to human settlements or inhabiting in areas considered for tourism could be facing a greater threat of infection. Fecal samples from Pygoscelis penguins (Pygoscelis spp.) were collected from different sites within Antarctic Peninsula and South Shetlands Islands in order to assess the presence of Salmonella enterica. Bacterial identification and characterization was performed applying biochemical and molecular techniques. Isolates were tested for antimicrobial resistance by the disk diffusion method, and PCR analyses were used for detection of resistance and virulence-associated genes. Four samples (1.74%) from P. papua were found to be positive to S. enterica serovar Enteritidis strains. All of them showed phenotypic antimicrobial resistance to at least three antimicrobials, and shared a similar gene profile through PCR. Results in this study urgently call for improvements in sanitary standards for waste disposal and sewage treatment in Antarctica. To our knowledge, this is the first study to report antimicrobial resistance in S. enterica isolated from Antarctic wild species.