Human skin aging is associated with functional deterioration on multiple levels of physiology, necessitating the development of effective skin senotherapeutics. The well-tolerated neurohormone melatonin unfolds anti-aging properties in vitro and in vivo, but it remains unclear whether these effects translate to aged human skin ex vivo. We tested this in organ-cultured, full-thickness human eyelid skin (5–6 donors; 49–77 years) by adding melatonin to the culture medium, followed by the assessment of core aging biomarkers via quantitative immunohistochemistry. Over 6 days, 200 µM melatonin significantly downregulated the intraepidermal activity of the aging-promoting mTORC1 pathway (as visualized by reduced S6 phosphorylation) and MMP-1 protein expression in the epidermis compared to vehicle-treated control skin. Conversely, the transmembrane collagen 17A1, a key stem cell niche matrix molecule that declines with aging, and mitochondrial markers (e.g., TFAM, MTCO-1, and VDAC/porin) were significantly upregulated. Interestingly, 100 µM melatonin also significantly increased the epidermal expression of VEGF-A protein, which is required and sufficient for inducing human skin rejuvenation. In aged human dermis, melatonin significantly increased fibrillin-1 protein expression and improved fibrillin structural organization, indicating an improved collagen and elastic fiber network. In contrast, other key aging biomarkers (SIRT-1, lamin-B1, p16INK4, collagen I) remained unchanged. This ex vivo study provides proof of principle that melatonin indeed exerts long-suspected but never conclusively demonstrated and surprisingly differential anti-aging effects in aged human epidermis and dermis.