Streptomyces sp. VITHM1 isolated from marine sediment sample collected at Alappuzha Beach, Kerala, India, was screened for antibacterial activity against Gram-positive and Gram-negative bacterial pathogens. The isolate was identified based on phylogeny and molecular taxonomic characterization. Bioactivity-guided extraction and purification of ethyl acetate extract of the isolate lead to identification of crystalline compound 2-hydroxy benzoic acid (HBA). HBA in methanol formed a needle-shaped crystal at room temperature after 36 h. Crystals formed were observed under scanning electron microscope for surface morphology, and the crystal structure was identified by using single-crystal X-ray diffraction analysis. The crystal was belonged to orthorhombic system, space group pbca with a= 11.631 (16) A, b= 7.1541 (8) A, c= 16.028 (2) A, V= 1334.2 (3) Å3. The molecular weight was calculated as 138.12 Daltons with a molecular formula C7 H6O3. The crystalline compound dissolved in ethyl acetate showed antibacterial activity against tested Gram-positive bacterial pathogens with the MIC value ranged from 12.5 to 25.0 μ g/mL and 12.5 to 75.0 μ g/mL against Gram-negative bacterial pathogens. HBA also exhibited concentration-dependent cytotoxicity on Vero cell line. This is the first report on extraction and identification of crystalline HBA from marine Streptomyces sp. VITHM1, and it can be explored further to be used as an effective antibacterial agent. © 2017, King Fahd University of Petroleum & Minerals.