Abstract Background Antibiotic therapy is one of the main methods of acne treatment; however, bacterial resistance is on the rise and can affect the treatment outcome. Quantitative bacteriologic cultures are the gold standard methodology for the assessment of such a problem; however, certain important biological aspects remain uncovered. Objective The purpose of this study was to compare the antibacterial activity of minocycline and lymecycline in sebaceous follicle infundibula and comedones of acne patients. Method We used a recently introduced flow cytometric method, allowing a distinction to be made between viable, injured (presumably resistant), and dead microorganisms. Results Minocycline (100 mg) proved to be superior to lymecycline (600 mg) in abating the microflora harboring in the sebaceous follicles of acne patients. Conclusions The dissimilar bioavailability and antimicrobial efficacy between the two bacteriostatic agents may impart different clinical efficacy.