The invention relates to the effective separation of compounds against acne inducing bacteria as anti-acne agents from leaves of Ocimum tenutflorum Linn. The invention also describes the process of separation of antimicrobial compounds using High Performance Thin Layer Chromatography (HPTLC) - Bioautographic technique, and identification of the separated antimicrobial active compounds by Gas Chromatography-Mass Spectrometry (GC-MS) analysis.