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Knowing that <i>Rhodomyrtus tomentosa</i> is known to have antibacterial effects, this study investigated the skin microbiota with a focus on <i>Cutibacterium acnes</i> (<i>C. acnes</i>) phylotypes in subjects with acne, and determined microbiota changes after 28 days of treatment with berries <i>Rhodomyrtus tomentosa</i> as an active ingredient (<i>RT</i>). Skin swabs from seventeen acne subjects were collected and the skin microbiome was analyzed using 16S rRNA gene sequencing. A culture-independent next-generation sequencing (NGS)-based SLST (single-locus sequence typing) approach was aimed at evaluating <i>RT</i> extract effects on <i>C. acnes</i> phylotype repartition. Clinical evaluations (lesion counts) were performed at baseline (D0) and after 28 days (D28) of twice-daily application of the <i>RT</i> active ingredient. We determined: (1) the skin microbiota at D0 was dominated by Actinobacteria followed by Firmicutes and Proteobacteria; (2) at the genus level, <i>Cutibacterium</i> was the most abundant genus followed by <i>Staphylococcus</i> and <i>Corynebacterium</i>; (3) <i>C. acnes</i> was the major species in terms of mean abundance, followed by <i>Staphylococcus epidermidis</i> (<i>S. epidermidis</i>) and <i>Staphylococcus hominis</i> (<i>S. hominis</i>); and (4) phylotype IA1 was most represented, with a predominance of SLST type A1, followed by phylotypes II, IB, IA2, IC, and III. After 28 days of <i>RT</i> extract treatment, phylotype repartition were modified with a decrease in abundance (approximately 4%) of phylotype IA1 and an increase in phylotype II and III. <i>Cutibacterium granulosum</i> (<i>C. granulosum</i>) abundance also decreased. Reduction of retentional and inflammatory lesions was also noted only after <i>RT</i> treatment; thus, <i>RT</i> extract acts as a microbiota-regulating agent.