Dental caries is a well known major oral health problem in most countries. Streptococcus mutans is recognized as the main pathogen of dental caries in human. This study was to assess the antimicrobial effectiveness of Chlorhexidine diacetate against oral microorganism. Ten samples were collected at dental clinics in and around Tirupur. All the isolates of S.mutans were identified by biochemically and five isolates were selected for antimicrobial activity. Chlorhexidine diacetate was prepared at different concentrations (0.05%, 0.10%, 0.20% and 0.25%) against S.mutans isolates. All the concentrations of CHX were showed the maximum inhibition in antimicrobial activity. This study one again proved that CHX is a gold standard against clinical oral pathogens.