<b>Background and Objective:</b> The RH3.5 was isolated from the rhizosphere of <i>Boesenbergia rotunda</i> (L.) Mansf. and identified to be <i>Streptomyces chartreusis</i> via analysis of its 16S rDNA sequence, chemotaxonomy and morphology. The aim of this study was to identify the major compounds of RH3.5 and assess their biological activities. <b>Materials and Methods:</b> Silica gel column chromatography and thin-layer chromatography were used to purify major compounds, elucidate 5,7,2'-trihydroxy-8-methoxyflavanone (compound <b>1</b>) and 5',2',5'-trihydroxy-7,8-dimethoxyflavanone (compound <b>2</b>). Subsequently, mass spectrometry and NMR techniques were used to identify the structure of these compounds. Antimicrobial, anti-inflammatory and cytotoxic properties were carried out using <i>in vitro</i> assays. <b>Results:</b> The bioassays revealed the antimicrobial effect of compounds <b>1</b> and <b>2</b> on MRSA and <i>Staphylococcus aureus</i>. The minimum inhibitory concentration and minimum bactericidal concentration was calculated in the range of 32-64 and 128-256 μg/mL, respectively. The compounds <b>1</b> and <b>2</b> also exhibited anti-inflammatory potential by inhibiting NO, IL-1β and TNF-α production in LPS-stimulated RAW264.7 cells in a dose-dependent manner. Additionally, they had mild cytotoxic action against Vero and L929 cell lines with IC<sub>50</sub> values greater than 512 μg/mL. <b>Conclusion:</b> These findings showed that flavonoids of <i>Streptomyces</i> <i>chartreusis</i> RH3.5 exhibited antibacterial and anti-inflammatory activities with low cytotoxicity against healthy cells. Thorough research on these compounds could result in the creation of useful methods for treating microbial infections and acute inflammatory responses.