Human hair follicles (HFs) constitute a unique microbiota habitat that differs substantially from the skin surface. Traditional HF sampling methods fail to eliminate skin microbiota contaminants or assess the HF microbiota incompletely, and microbiota functions in human HF physiology remain ill explored. Therefore, we used laser-capture microdissection, metagenomic shotgun sequencing, and FISH to characterize the human scalp HF microbiota in defined anatomical compartments. This revealed significant compartment-, tissue lineage-, and donor age-dependent variations in microbiota composition. Greatest abundance variations between HF compartments were observed for viruses, archaea, Staphylococcus epidermidis, Cutibacterium acnes, and Malassezia restricta, with the latter 2 being the most abundant viable HF colonizers (as tested by propidium monoazide assay) and, surprisingly, most abundant in the HF mesenchyme. Transfection of organ-cultured human scalp HFs with S. epidermidis-specific lytic bacteriophages ex vivo downregulated transcription of genes known to regulate HF growth and development, metabolism, and melanogenesis, suggesting that selected microbial products may modulate HF functions. Indeed, HF treatment with butyrate, a metabolite of S. epidermidis and other HF microbiota, delayed catagen and promoted autophagy, mitochondrial activity, and gp100 and dermcidin expression ex vivo. Thus, human HF microbiota show spatial variations in abundance and modulate the physiology of their host, which invites therapeutic targeting.