Lactobacillus fermentum CECT 5716, isolated from human milk, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays, which suggests a strong potential as a probiotic strain. In this work, some phenotypic properties of L. fermentum CECT 5716 were evaluated, and the genetic basis for the obtained results was searched for in the strain genome. L. fermentum CECT 5716 does not contain plasmids and showed neither bacteriocin nor biogenic amine biosynthesis ability but was able to produce organic acids, glutathione, riboflavin, and folates and to moderately stimulate the maturation of mouse dendritic cells. No prophages could be induced, and the strain was sensitive to all antibiotics proposed by European Food Safety Authority (EFSA) standards, while no transmissible genes potentially involved in antibiotic resistance were detected in its genome. Globally, there was an agreement between the phenotype properties of L. fermentum CECT 5716 and the genetic information contained in its genome.