The present study deals with the synthesis of titanium dioxide (TiO2) nanoparticles using Pandanus odorifer (Forssk.) Kuntze leaf extract and explores the antimicrobial potential of synthesized nanoparticles. The biosynthesized TiO2 nanoparticles were characterized using different spectroscopic and microscopic techniques. The absorption spectrum of synthesized TiO2 nanoparticles was primarily characterized by Ultraviolet-visible (UV–vis) spectrophotometer. The functional groups associated with the TiO2 nanoparticles and the Pandanus odorifer leaf extract were examined by Fourier Transform Infrared (FTIR) spectroscopy. The crystalline structure of nanoparticles was analyzed by X-ray diffraction (XRD) examination. Morphological characters were examined by Scanning Electron Microscopy (SEM). Presence of elemental composition of synthesized TiO2 nanoparticles was characterized by Energy Dispersive X-ray (EDX). The antimicrobial properties of the TiO2 nanoparticles were observed to be highly toxic against bacterial strains are Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) , and the fungal strains are Aspergillus niger (A. niger), Candida albicans (C. albicans). The zone of inhibition was estimated by disc diffusion assay and moreover, minimum inhibitory concentration was evaluated by the Resazurin Microtitre Assay. It can be concluded that titanium dioxide nanoparticles manifest a strong antimicrobial action and thus can be developed as a novel type of a antimicrobial material for the cure of microbial infections.