Vitamins B6 (i.e., pyridoxin and its analogues) and B7 (i.e., biotin or vitamin H) are essential molecules for many physiological processes. In addition to their well-known involvement in several enzymatic reactions, recent discoveries revealed their participation in other processes, e.g. in gene expression via epigenetic processes, including biotinylation of proteins in the case of biotin. Plants, fungi, archaea and most bacteria synthesize both vitamins, whereas animals and humans lack enzymes for their biosynthesis and depend on their exogenous supply. At least in case of biotin, human gastrointestinal microbiota can likely partly satisfy the need. Both vitamins are water soluble and require a transporter for efficient absorption after oral administration; they can be rapidly excreted, hence being considered largely non-toxic. In addition to physiological and kinetic aspects of vitamins B6 and biotin, this review, which is based on a search in PubMed up to 2023, covers sources of these vitamins, the impact of food treatment on their content, causes and symptoms of deficiency and specific mutations related to their function. Currently available literature on the analytical determination of these vitamins in biological fluids, possible pharmacological uses, and symptoms of toxicity, although rare, are also included.