Background: There are two main strategies to eliminate Cutibacterium acnes (C. acnes) and to reduce antibiotic resistance in acne treatment. The first is to target the pathogenic bacteria and the second is to change the environment for their growth. Aim: The present study aimed to evaluate the anti-microbial role of non-antibiotic agents against C. acnes in acne vulgaris patients. The three agents of interest in the study were isotretinoin, salicylic acid, and N-acetylcysteine (NAC). Methods: The study included forty-eight patients with acne vulgaris with ages ranging from 16 to 30 years, and they had different grades of the disease. Azithromycin and Doxycycline sensitivity and the ability of biofilm formation of C. acnes isolated from all patients were assessed before and after adding the 3 agents. Results: Azithromycin and Doxycycline sensitivity was improved after adding the 3 agents and the ability of biofilm formation of C. acnes was also reduced. Conclusions: Isotretinoin, salicylic acid, and NAC can be promising adjuvants in treating acne vulgaris by their anti-microbial effect in reducing biofilm formation and improving antibiotic sensitivity.