Investigates the microbial communities of plants and Veggie pillow components with culturable and non-culturable methods providing qualitative and quantitative data. This studies also stresses the food safety component of fresh crops grown on the ISS. The VEG-03D technology demonstration was completed achieving simultaneous multi-species plant growth within a Veggie unit on the International Space Station (ISS). Microbiological characterization using molecular and culture-based methods was performed on leaves and roots from two harvests of three leafy greens, red romaine lettuce (Lactuca sativa cv. ‘Outredgeous’); mizuna mustard, (Brassica rapa var japonica); and green leaf lettuce, (Lactuca sativa cv. Waldmann’s) and associated rooting pillow components and Veggie chamber surfaces. Culture based enumeration and pathogen screening indicated the leafy greens were safe for consumption. Surface samples of the Veggie facility and plant pillows revealed low counts of bacteria and fungi and are commonly isolated on ISS. Community analysis was completed with 16S rRNA amplicon sequencing. Comparisons between pillow components, and plant tissue types from VEG-03D, E, and F revealed higher diversity in roots and rooting substrate than the leaves and wick. This work provides valuable information for food production-related research on the ISS and the impact of the plant microbiome on this unique closed environment.