The present study was conducted to evaluate the antimicrobial activity of ethanolic root extract of Limonia acidissima against specific organism like gram negative, gram positive and yeast. The well diffusion assay technique was adopted for the antimicrobial activity. The plant extract 50 µl was used and the streptomycin 10 µg was used as reference control. The plant extract of Limonia acidissima exhibited significant antimicrobial activity by inhibiting the microbial growth by sufficient zone of inhibition and the effect was comparable with reference control. From the result it was concluded that ethanolic root extract of Limonia acidissima possesss antimicrobial activity.