Many medicinal plants have been used for centuries in daily life to treat microbial diseases all over the world. In this study, the in vitro antibacterial activity of aqueous and ethanol root extracts of Thespesia populnea Linn were investigated. Antimicrobial properties of T. populnea Linn was evaluated against five pathogenic bacteria and two fungi. Disc diffusion method and minimum inhibitory concentration (MIC) were determined by broth serial dilution method. The ciprofloxacin (5 μg/ml) and flucanozole (100 units/disc) were used as positive controls for bacteria and fungi respectively. Different concentrations (50, 100, 150 μg/ml) of ethanolic and aqueous root extracts of T. populnea were checked for the dose dependent antibacterial activity. Thespesia populnea showed broad spectrum antimicrobial activity against gram positive and gram negative bacteria and maximum inhibition by ethanolic extract was observed at higher dose (250 μg/ml) as 27±0.2mm. The MIC of the ethanol extract was 10 μg/ml for Staphylococcus aureus and 750 μg/ml for Candida albicans. The antifungal activity offered against S. aureus by the ethanolic extract is more than the aqueous extract. The results concluded that the anti-microbial activity of T. populnea was dose dependent. As the concentration increased the inhibition zone also increased. Flavonoids and tannins present in the extracts may be responsible for the antimicrobial activity.