Introduction: Changes in the skin microbiome are associated with acne vulgaris (AV), a condition characterized by comedones, papules, and pustules. While some bacteria have been studied, many others remain unexplored, highlighting the need to understand the microbiome differences between acne-prone and normal skin. This study aimed to compare skin microbiome profiles between AV patients and healthy individuals and to explore associations with microbial diversity and specific bacterial populations involved in AV pathogenesis. Methods: A total of 144 participants were recruited, comprising 36 AV patients and 108 healthy controls. Skin samples were collected from the left cheek after standardized preconditioning. DNA was extracted using the DNeasy PowerSoil Kit™, and the V3–V4 region of 16S rRNA was amplified and sequenced. Microbial diversity was assessed by the Shannon index, and correlations with sebum levels were analyzed. Results: The results revealed significant differences in microbial diversity, with AV patients exhibiting a markedly lower Shannon index compared to controls, indicating decreased microbial diversity and potential dysbiosis. While the relative abundance of Cutibacterium acnes, a bacterium commonly associated with AV, showed no significant differences between the two groups, the prevalence of Staphylococcus epidermidis was notably higher in AV patients. This suggests that S. epidermidis may play a complex role in the inflammatory processes associated with AV. Moreover, the study identified a negative correlation between microbial diversity and sebum levels, suggesting that increased sebum production may favor the growth of S. epidermidis, potentially exacerbating the condition. Conclusion: These findings highlight the interaction between host factors and microbial composition. This study emphasizes the role of skin microbiome dysbiosis in acne vulgaris and provides insights for future microbiome-based therapeutic strategies. Further research is needed to clarify microbial mechanisms and potential interventions targeting the microbiome in the management of acne vulgaris. © 2026 Sitohang et al.