The International Space Station and the Antarctic Research Station Concordia are confined habitats in extreme environments. The human and habitat microflora can alter due to the special environmental conditions resulting in contamination and health risk for the crew. In this study, 29 isolates from the ISS and 55 from Concordia belonging to the genera Staphylococcus and Enterococcus were investigated. Resistance to one or more antibiotics was detected in 75.8% of the ISS and in 43.6% of the Concordia strains. The corresponding resistance genes were identified by polymerase chain reaction in 86% of the resistant ISS strains and in 18.2% of the resistant Concordia strains. Plasmids are present in 86.2% of the ISS and in 78.2% of the Concordia strains. Relaxase and/or transfer genes encoded on plasmids from gram-positive bacteria like pIP501, pRE25, pSK41, pGO1 and pT181 were detected in 86.2% of the ISS and in 52.7% of the Concordia strains. Most pSK41-homologous transfer genes were detected in ISS isolates belonging to coagulase-negative staphylococci. Biofilm formation was observed in 83% of the ISS and in 92.7% of the Concordia strains. In conclusion, ISS isolates were shown to encode more resistance genes and possess a higher gene transfer capacity than Concordia isolates.