A new biologically active Allelochemical 3,5,7,3’,4’-pentahydroxyflavone-3<em>-O-β</em>-<em>D </em>xylopyranosyl -(1→4)- <em>β-D</em>-glucopyranosyl-4<sup>’</sup>-<em>O-α-</em>L-arabinoside (<strong>A</strong><sub>1</sub><strong>) </strong>whose m.p. is 340–345 <sup>0</sup>C, m.f. [M<sup>+</sup>] 728 (EIMS) was isolated from methanolic leaf extracts of<strong> </strong><em>Dalbergia paniculata</em> Roxb. It was characterized by several color reactions, spectral analysis FTIR, <sup>1</sup>H-NMR, GC-MS, and chemical degradations. The results of the antimicrobial activity of <strong>(A<sub>1</sub>) </strong>at high concentration (100µg/mL) was 20, 17, and 15 mm zone of inhibition against <em>E. coli</em>, <em>B. cereus, </em>and <em>S. aureus aeruginosa, </em>respectively. The data demonstrated that the antimicrobial activity of CH<sub>3</sub> OH soluble fraction <strong>(A<sub>1 </sub>) </strong>at all concentrations was highest against <em>E. coli</em> and lowest<em> S. aureus.</em> <em>E. coli</em>, <em>B. cereus, </em>and <em>S. aureus aeruginosa, </em>respectively 7.3, 2.1, and 0.02mm, MIC was defined as the minimum concentration of assayed samples that inhibited the visible growth of the tested microorganism<em>. </em>MIC in <em>E. coli</em>, <em>B. cereus, </em>and <em>S. aureus aeruginosa </em>was 7.3, 2.1, and 0.02mm, respectively.