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In this comprehensive study, the synthesis of titanium dioxide (TiO2) was meticulously performed utilizing Ficus religiosa’s aqueous bark extract, commonly known as the Peepal tree. Employing a green approach, the resulting nanoparticles displayed a distinctive green hue, which was discerned through a series of advanced analytical techniques including TEM (Transmission electron microscopy), UV–visible spectroscopy, XRD (X-ray diffraction), AFM (Atomic force microscopy), SEM–EDX (Scanning Electron Microscopy- Energy Dispersive X-Ray Spectrometry), and FT-IR (Fourier transform infrared spectroscopy). The characterization revealed that the greenly synthesized titanium dioxide nanoparticles exhibited a diverse range of sizes and shapes, further highlighting the effectiveness of the eco-friendly synthesis approach. Subsequently, the antibacterial characteristics of these nanoparticles were rigorously evaluated against both Gram-negative & Gram-positive bacteria using the agar disc diffusion method. Notably, a pronounced zone of inhibition was observed against E. coli(Gram-negative bacteria) compared to S. aureus(Gram-positive bacteria), suggesting a potential variation in antibacterial efficacy against different bacterial strains. Overall, this study represents a pioneering effort in the field, emphasizing the utilization of a green synthesis approach for the precise preparation of TiO2nanoparticles from Ficus religiosa bark extract, while concurrently investigating their antibacterial activity, thereby contributing significantly to the advancement of eco-friendly nanoparticle synthesis methodologies.