Background. Although several energy devices targeting sebaceous glands have been developed, an effective and safe therapeutic tool for hyperseborrhea is still needed. Nonthermal atmospheric-pressure plasma (NTAPP) induces microscopic tissue reactions in sebaceous glands of rat skin. Objective. The aim of the study is to investigate the effects of NTAPP on sebum production in human skin in vivo followed by an experimental study of human sebocytes. Methods. Fourteen healthy volunteers with oily facial skin underwent three sessions of argon- and nitrogen-NTAPP treatment at a 1-week interval and were followed up for 8 weeks. The casual sebum level, sebum excretion rate, and porphyrin index were evaluated. Histological analysis was performed using skin biopsy specimens taken from two subjects at the baseline and week 2. SZ95 sebocytes were stimulated with testosterone and linoleic acid (T/LA) with or without treatment with NTAPP. BODIPY and Nile red staining were used for qualitative lipids analysis. Proliferation and differentiation markers were also assessed. Results. Casual sebum levels and sebum excretion rates in facial skin decreased by 26 and 24%, respectively, at week 4 compared to those of the baseline. Porphyrin index also decreased by 38% at week 2. Histologically, NTAPP-treated human skin showed no obvious thermal injury, but the number of Ki67+ cells in the sebaceous glands decreased at week 2. Argon- and nitrogen-NTAPP attenuated T/LA-induced increases in neutral lipid accumulation, Ki67+ cells, and peroxisome proliferator-activated receptor-ɣ transcription in human sebocytes at energy settings that did not induce apoptosis. Conclusion. Argon- and nitrogen-NTAPP can be a safe and effective therapeutic tool for hyperseborrhea-associated diseases such as acne. This trial is registered with NCT04917835.