Fungi serves a significant purpose in matter circulation because of their ability to decompose complex substances into simpler compounds. Strict isolation facilities, such as the ISS or AATC Lunar Habitat were forced to develop efficient waste-management solutions, in order to generate as little waste as possible. It was hypothesized that P. otreatus mycelium enriched with silver nanoparticles (AgNPs), which possess biocidal properties, can be included as a controller of an organic matter recirculation system. The aim of the experiment was to examine the impact of AgNPs on P. ostreatus mycelium growth, as well as to determine if AgNPs can protect P. ostreatus mycelium cultures against pathogens. P. ostreatus mycelium cultivation was established onboard AATC Analog Lunar Habitat in Rzepiennik Strzyżewski, Poland. It was observed that P. ostreatus mycelium cultures (both prepared in a laminar cabinet and a glove box) developed properly in the environment of the Analog Habitat. AgNPs visibly inhibited mycelium growth in comparison to control. P. ostreatus showed culturing potential in the Analog Habitat conditions. Thus, once a proper amount is harvested, it can be used in organic matter recirculation system. The efficiency of the system may be increased due to AgNPs addition, serving as anti-microbial substances.