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Biofilms of <i>Cutibacterium</i> (<i>C.</i>) <i>acnes</i> (formerly <i>Propionibacterium acnes</i>) are responsible for the persistence and antibiotic resistance of acne vulgaris. In addition to the standard treatments for acne vulgaris, a common adjunctive treatment is the topical administration of nicotinamide (NAM). However, the effects of NAM on biofilms of <i>C. acnes</i> have never been explored. This study comprehensively investigates the effects of NAM against biofilms of <i>C. acnes</i> using in vitro and in vivo approaches. The results showed that NAM potentiated the efficacy of suboptimal dosing of tetracycline against <i>C. acnes</i>. Moreover, NAM alone decreased the formation and increased the degradation of biofilms in <i>C. acnes</i>. The antibiofilm effect of NAM against <i>C. acnes</i> was further enhanced in combination with deoxyribonuclease (DNase) I, an enzyme with known antibiofilm properties. The computational molecular docking, surface plasmon resonance analysis, and enzymatic kinetic assay demonstrated that NAM binds to DNase I and accelerated its reaction. In conclusion, NAM activates DNase I to attenuate biofilms of <i>C. acnes</i>. This offers valuable insights into the strategies against biofilms that are worth elaborating on in other biofilm-related chronic cutaneous infections in the future.