Background Staphylococcus aureus is known to form a biofilm and colonize on damaged skin of the hands. We investigated changes in the quantity of S aureus on the hands and changes in skin damage when using a hand-cleansing formulation with potassium oleate but without a sanitizer (formulation A), which is highly effective in removing S aureus biofilm and causes minimal skin damage. Material and Methods The participants (14 medical staff members) used 2 types of hand-cleansing formulations (formulations A and B), each for 4 weeks. S aureus of the hands was cultured from swab samples on agar plates. Surface of hands was measured using an ultraviolet light microscope. Results and Discussion The quantity of S aureus after using formulation A for 4 weeks was 101.08 ± 0.05 CFU/mL, a statistically significant decrease from the quantity of S aureus (101.59 ± 0.19  CFU/mL) just before use ( P  = .029). Also, dryness of hand surfaces decreased. With formulation B, the quantity of S aureus did not significantly change from before to after use ( P  > .05). This presumably occurs because formulation A gently removes S aureus biofilm. Conclusions Formulation A removed S aureus from the hands of participants, and skin damage on the hands improved.