This article describes a green and sustainable approach for the synthesis of reduced graphene oxide (rGO) using Indigofera tinctoria leaf extract as a natural reducing agent. The biogenic IT-rGO was characterized using UV-Vis spectroscopy, FTIR, XRD, and SEM to confirm its successful reduction and to determine its structural properties. The phytochemicals present in the extract, including flavonoids, polyphenols and alkaloids, played a crucial role in reducing GO to rGO. The synthesized rGO exhibited significant antibacterial activity against Escherichia coli, with the highest antibacterial activity observed at a 100% IT-rGO concentration, resulting in an inhibition zone value of 10.25 mm against the E. coli bacteria. The antibacterial mechanism is proposed to involve membrane disruption and the induction of oxidative stress. The use of Indigofera tinctoria not only offers an eco-friendly and cost-effective route for rGO production but also imparts enhanced bioactivity, making the material a promising candidate for biomedical and environmental antibacterial applications.