Objective: This study was conducted to identify some bioactive compounds of Boesenbergia rotunda rhizome and to test as antioxidant and antimicrobial agents. Methods: The milled dried rhizome of B. Rotunda (5kg) was extracted exhaustively with ethanol. The ethanol extract was partitioned three times by n-hexane, chloroform, and ethyl acetate respectively. Each fraction was fractionated by vacuum liquid chromatography (VLC) and then purified by column chromatography gravitation. Structural identification of all pure compounds were elucidated based on spectroscopic methods (UV, IR, and NMR).The antioxidant activity was tested by 2,2-diphenyl- 1-picrylhydrazyl (DPPH). Antimicrobial character was screened for activities againts pathogenic bacteria i.e. Escherichia coli ATCC-11229, Staphylococcus aureus ATCC-25923, Staphylococcus epidermidis FNCC-0048, and Streptococcus mutans by the disk-diffusion method. The assay was done in triplicate, and chloramphenicol was used as the positive control. Results and Discussion: From ethanol extract of B. rotunda three known compounds of flavanones, namely 2’,4’-dihydroxy-6-methoxychalcone (compound-1), 5-hydroxy-7- methoxyflavanone (compound-2), and 5,7-dihydroxyflavanone (compound-3) were isolated. The ethanol extract of B. rotunda and the three isolated compounds -1,-2, and -3 showed an antioxidant activity with the IC value of 92.64; 46.66; 62.84, and 62.66 μg/mL, respectively. The zone of inhibition of extract and the three isolated compounds showed moderate activity against Escherichia coli ATCC-11229, Staphylococcus aureus ATCC-25923, Staphylococcus epidermidis FNCC-0048, and Streptococcus mutans.The maximum zone of inhibition was 13.20±0.76 mm at the maximum concentration used (500 μg/mL) against Escherichia coli ATCC-11229, and the minimum inhibitory concentration(MIC) for each bacteria was found to be 0.5μg/mL. Conclusion: The result of the study suggests that B. rotunda rhizome contains potential bioactive compounds which could be suitable for antioxidant and the treatment of various infections caused by Escherichia coli ATCC-11229, Staphylococcus aureus ATCC- 25923, Staphylococcus epidermidis FNCC-0048, and Streptococcus mutans.