The skin tissue of the scalp is unique from other skin tissues because it coexists with hair, and many differences in microbial composition have been confirmed. In scalp tissues, hair loss occurs due to a combination of internal and external factors, and several studies are being conducted to counteract this. However, not many studies have addressed hair loss from the perspective of the microbiome. In this study, subjects with hair loss and those with normal scalps were set as experimental and control groups, respectively. In the experimental group, hair loss had progressed, and there was a large difference in microbiome composition compared to the group with normal scalps. In particular, differences in Accumulibacter, Staphylococcus, and Corynebacterium were found. From Staphylococcus epidermidis Cicaria, two active components were isolated as a result of repeated column chromatography. Spectroscopic data led to the determination of chemical structures for adenosine and biotin. Fractions were obtained, and ex vivo tests were conducted using hair follicles derived from human scalp tissue. When the microbiome adenosine-treated group was compared to the control group, hair follicle length was increased, and hair root diameter was maintained during the experimental periods. In addition, the Cicaria culture medium and the microbial adenosine- and biotin-treated groups maintained the anagen phase, reducing progression to the catagen phase in the hair growth cycle. In conclusion, it was confirmed that the Cicaria culture medium and the microbial adenosine and biotin derived from the culture were effective in inhibiting hair loss.