Microbes are present in all occupied indoor environments, including the International Space Station (ISS). An unexpected ventilation system failure may lead to microbial and fungal growth that is associated with material degradation or negative health effects. There is a need for improved understanding of fungal function when exposed to moisture in indoor spaces. A de novo metatranscriptomic study was performed using dust collected from within the ISS. After returning to Earth, dust was incubated in laboratory chambers to simulate relative humidity conditions of 50%, 85%, or 100% equilibrium relative humidity (ERH) for 1-week. Changes in fungal function (gene expression) were associated with moisture exposure. Genes associated with secondary metabolism and fungal growth were significantly upregulated (p ≤ 0.001, log2FC ≥ 2) at elevated RH conditions (85% or 100% ERH). Elevated moisture conditions showed an increased expression of mycotoxin and fungal allergen genes such as Asp f 4 and Alt a 7 (log2FC ≥ 5). These results demonstrate that understanding microbial gene expression in response to elevated moisture will help improve microbial monitoring standards for confined spacecraft environments and help protect astronaut health and spacecraft integrity.