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The aim of the work is to synthesize iron oxide (α-Fe2O3) nanoparticles using Sida cordifolia plant extract along with evaluation of its antibacterial activity. The presence of phytochemicals in Sida cordifolia methanolic plant extract was investigated by HPTLC and LC-MS/TOF. The probable mechanism for formation of α-Fe2O3 nanoparticles in mediation with plant extract was demonstrated. The green synthesized iron oxide nanoparticles (α-Fe2O3 NPs) were characterized by using X-ray diffraction, scanning, and transmission electronic microscopy, TG-DTA, FTIR, and UV spectroscopy. The crystallite size of prepared α-Fe2O3 nanoparticles estimated via Debye-Scherrer formula and Williamson-Hall plot was around 20 nm which is in accordance with particle size in TEM images. The S. cordifolia mediated iron-oxide nanoparticles (α-Fe2O3 NPs) hold potent antibacterial activity against various gram positive and gram negative bacteria.