The ability to grow safe, fresh food to supplement packaged foods of astronauts in space has been an important goal for NASA. Food crops grown in space experience different environmental conditions than plants grown on Earth (e.g., reduced gravity, elevated radiation levels). To study the effects of space conditions, red romaine lettuce, Lactuca sativa cv ‘Outredgeous,’ plants were grown in Veggie plant growth chambers on the International Space Station (ISS) and compared with ground-grown plants. Multiple plantings were grown on ISS and harvested using either a single, final harvest, or sequential harvests in which several mature leaves were removed from the plants at weekly intervals. Ground controls were grown simultaneously with a 24–72 h delay using ISS environmental data. Food safety of the plants was determined by heterotrophic plate counts for bacteria and fungi, as well as isolate identification using samples taken from the leaves and roots. Molecular characterization was conducted using Next Generation Sequencing (NGS) to provide taxonomic composition and phylogenetic structure of the community. Leaves were also analyzed for elemental composition, as well as levels of phenolics, anthocyanins, and Oxygen Radical Absorbance Capacity (ORAC). Comparison of flight and ground tissues showed some differences in total counts for bacteria and yeast/molds (2.14 – 4.86 log10 CFU/g), while screening for select human pathogens yielded negative results. Bacterial and fungal isolate identification and community characterization indicated variation in the diversity of genera between leaf and root tissue with diversity being higher in root tissue, and included differences in the dominant genera. The only difference between ground and flight experiments was seen in the third experiment, VEG-03A, with significant differences in the genera from leaf tissue. Flight and ground tissue showed differences in Fe, K, Na, P, S, and Zn content and total phenolic levels, but no differences in anthocyanin and ORAC levels. This study indicated that leafy vegetable crops can produce safe, edible, fresh food to supplement to the astronauts’ diet, and provide baseline data for continual operation of the Veggie plant growth units on ISS.