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<i>Cutibacterium acnes</i> (<i>C. acnes</i>) is the main causative agent of acne vulgaris. The study aims to evaluate the antimicrobial activity of a natural product, <i>Arctostaphylos uva-ursi</i> leaf extract, against <i>C. acnes</i>. Preliminary chemical–physical characterization of the extract was carried out by means of FT-IR, TGA and XPS analyses. Skin permeation kinetics of the extract conveyed by a toning lotion was studied in vitro by Franz diffusion cell, monitoring the permeated arbutin (as the target component of the extract) and the total phenols by HPLC and UV-visible spectrophotometry, respectively. Antimicrobial activity and time-killing assays were performed to evaluate the effects of <i>Arctostaphylos uva-ursi</i> leaf extract against planktonic <i>C. acnes</i>. The influence of different <i>Arctostaphylos uva-ursi</i> leaf extract concentrations on the biofilm biomass inhibition and degradation was evaluated by the crystal violet (CV) method. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was used to determine the viability of immortalized human keratinocytes (HaCaT) after exposure to <i>Arctostaphylos uva-ursi</i> leaf extract for 24 and 48 h. Levels of interleukin (IL)-1β, IL-6, IL-8 and tumour necrosis factor (TNF)-α were quantified after HaCaT cells cotreatment with <i>Arctostaphylos uva-ursi</i> leaf extract and heat-killed <i>C. acnes</i>. The minimum inhibitory concentration (MIC) which exerted a bacteriostatic action on 90% of planktonic <i>C. acnes</i> (MIC₉₀) was 0.6 mg/mL. Furthermore, MIC and sub-MIC concentrations influenced the biofilm formation phases, recording a percentage of inhibition that exceeded 50 and 40% at 0.6 and 0.3 mg/mL. <i>Arctostaphylos uva-ursi</i> leaf extract disrupted biofilm biomass of 57 and 45% at the same concentrations mentioned above. Active <i>Arctostaphylos uva-ursi</i> leaf extract doses did not affect the viability of HaCaT cells. On the other hand, at 1.25 and 0.6 mg/mL, complete inhibition of the secretion of pro-inflammatory cytokines was recorded. Taken together, these results indicate that <i>Arctostaphylos uva-ursi</i> leaf extract could represent a natural product to counter the virulence of <i>C. acnes,</i> representing a new alternative therapeutic option for the treatment of acne vulgaris.