Antibiotic-resistant Cutibacterium acnes and dysbiosis of the skin microbiome are of increasing concern in acne treatment. Enterococcus faecalis, a widely used probiotic, has shown benefits for acne treatment by exerting antimicrobial activity against C. acnes. Therefore, this study aimed to investigate the efficacy and safety of an E. faecalis CBT SL-5-extract-containing lotion in patients with mild-to-moderate acne. Twenty patients were enrolled in this randomized, placebo-controlled, split-face comparative study. Patients were treated with E. faecalis lotion on one side of the face and a vehicle lotion on the other side for 4 weeks. The efficacy outcome measures included improvement in the investigators’ assessment of acne severity, patient satisfaction, changes in skin parameters and diversity of the skin microbiome. The investigators’ assessment score was significantly improved on the test side compared to the control side, after 2 weeks (p = 0.009) and 6 weeks (p < 0.0005). However, TEWL and skin hydration were not significantly different between the two groups. The phylogenetic diversity of the skin microbiota decreased over time in the skin samples of test side. In conclusion, E. faecalis CBT SL-5 extract can be a feasible and well-tolerated option for improving acne severity and skin microbiome dysbiosis in mild-to-moderate acne patients.