Background: Screening of natural compounds for the development of anti-acne therapeutic agents has been steadily required considering various side effects of acne medications. However, previous studies have mainly focused on experimental tests without clinical trials and histopathological analysis. Objectives: To compare the clinical efficacy, safety and histopathological changes between Lactobacillus-fermented Chamaecyparis obtusa (LFCO) and existing tea tree oil (TTO). Methods: A total of 34 patients were instructed to apply 5% LFCO to the involved areas of a randomly allocated side and 5% TTO extract to the other side for 8 weeks in a double-blind split-face clinical trial. Results: After 8 weeks, inflammatory acne lesions were reduced by 65.3% on the LFCO side and by 38.2% on the TTO side. LFCO was also superior to TTO in the onset time of efficacy (p < 0.05). The LFCO side further demonstrated improvement for non-inflammatory lesions (52.6%, p < 0.05), decreased size of sebaceous glands and sebum output reductions. Patients' subjective satisfaction was also higher without severe adverse reactions. Protein expressions of nuclear factor κB decreased earlier on the LFCO side, and those of interleukin-1a (IL-1a), IL-8, insulin-like growth factor 1 receptor and sterol regulatory element-binding protein 1 decreased subsequently. Ultra-performance liquid chromatography/high-resolution mass spectrometry further demonstrated that the contents of dihydroxybenzoic acid, taxifolin and quercetin were increased in LFCO after fermentation. Conclusions: LFCO treatment was rapid and effective for treating acne lesions compared to TTO. Histopathological findings correlated well with the clinical acne grade and treatment response. This novel natural compound appears to be effective and safe for acne treatment.