One of the biggest challenges of science is the determination of whether extraterrestrial life exists. Although potential habitable areas might be available for complex life, it is more likely that microbial life could exist in space. Many extremotolerant and extremophilic microbes have been found to be able to withstand numerous, combined environmental factors, such as high or low temperatures and pressures, high-salt conditions, high doses of radiation, desiccation or nutrient limitations. They may even survive the transit from one planet to another. Terrestrial Mars-analogue sites are one focus of researchers, in order to understand the microbial diversity in preparation for upcoming space missions aimed at the detection of life. However, such missions could also pose a risk with respect to contamination of the extraterrestrial environment by accidentally transferred terrestrial microorganisms. Closer to the Earth, the International Space Station is the most enclosed habitat, where humans work and live—and with them numerous microorganisms. It is still unknown how microbes adapt to this environment, possibly even creating a risk for the crew. Information on the microbiology of the ISS will have an impact on the planning and implementation of long-term human spaceflights in order to ensure a safe, stable and balanced microbiome on board.