The antimicrobial activity of the methanolic extract of the bark of Holarrhena pubescens, its fractions, and conessine, a steroidal alkaloid, was determined against various bacteria and fungi using the agar diffusion method. They were all found to possess significant activity against some of the bacteria tested. The alkaloidal fraction and conessine also exhibited marginal activity against some of the fungi tested. The minimum inhibitory concentration (MIC) value of conessine was determined against various bacteria, and the highest activity was seen against Micrococcus luteus ATCC 9341 (MIC: 15.6 µg per disc).