The background of this study chlorhexidine gluconate is an effective bactericidal agent and broad-spectrum antimicrobial drug. It has been extensively researched and is the “gold standard” antimicrobial in oral hygiene. Among different kind of microorganisms the cariogenic Lactobacillus acidophilus is recognized as a predominant pathogen in childhood caries. So plaque samples were collected from different dental clinics. The isolates were identified by biochemical characterization and 16SrDNA gene sequencing. Further, the antimicrobial production was assessed. Lactic acid production by L. acidophilus is partially growth associated followed by quantification of organic acid and determination of pH value indicates that organic acid production was increased with the highest acidity (1.8%) and determination of H₂O₂ production by the specific 5 isolates generated H₂0_2. The highest proportion was found 5 out of 5 strain (100%) being positive. Finally antimicrobial efficacy of CHX was used at 2% concentration (50µl, 100µl and 150µl) against L. acidophilus.