Genomic and proteomic characterization of the Aspergillus niger isolate, JSC-093350089, collected from U.S. segment surfaces of the International Space Station (ISS) is reported, along with a comparison to the experimentally established strain ATCC 1015. Whole-genome sequencing of JSC-093350089 revealed enhanced genetic variance when compared to publicly available sequences of A. niger strains. Analysis of the isolate’s proteome revealed significant differences in the molecular phenotype of JSC-093350089, including increased abundance of proteins involved in the A. niger starvation response, oxidative stress resistance, cell wall integrity and modulation, and nutrient acquisition. Together, these data reveal the existence of a distinct strain of A. niger onboard the ISS and provide insight into the molecular phenotype that is selected for by melanized fungal species inhabiting spacecraft environments.