Sustainable agriculture in microgravity is integral to future long-term space exploration. To ensure the efficient and sustainable cultivation of plants in space, a contingency plan to monitor plant health and mitigate plant diseases is necessary. Yet, neither methods nor tools currently exist to evaluate the plant microbial interactions or to diagnose potential plant diseases in space-based bioregenerative life support systems (BLSS). Here we show how the MinION sequencing platform can be used to diagnose the opportunistic pathogen, Fusarium oxysporum; a fungal infection on Zinnia hybrida plants that were grown on the International Space Station (ISS) in 2015 (Schuerger et al., 2021). Genomic DNA from the infected plant material (root and leaf tissues) retrieved from the ISS were extracted and sequenced. In addition, pure cultures of Burkholderia contaminans, Fusarium oxysporum, and Fusarium sporotrichioides were used as controls to test the specificity of the bioinformatics pipeline developed. The results show that the MinION can be used to accurately differentiate between fusarium species and strengthens the case for using the system as a rapid plant disease diagnostic tool.