Nanoparticles offer exciting potential roles in targeted drug delivery mechanisms, allowing for precise and controlled release of medications into particular cells or tissues with minimal adverse effects. This study examined the antimicrobial activity of green-synthesised titanium nanoparticles from Prosopis cineraria leaf extract and determined their bactericidal properties by means of a live/dead assay. The bio-synthesized titanium dioxide (TiO₂) nanoparticles showed a peak at 2θ values of 25.52° in X-ray diffraction, while scanning electron microscopy revealed it to be smooth and irregular in shape. Energy dispersive X-ray spectroscopy analysis of TiO₂ nanoparticles calcined at 550 °C confirmed the presence of only titanium and oxygen, with no detectable impurities. Atomic and weight percentages of titanium and oxygen were 23.54% and 47.96%, and 76.46% and 52.04%, respectively. The bio-synthesized TiO₂ nanoparticles was assessed for antimicrobial activity against bacterial strains and fungal isolates. The Titanium oxide‚ NPs exhibited effective antibacterial activity, with Pseudomonas aeruginosa showing the highest inhibition zone (13 mm), followed by Escherichia coli (12 mm), Staphylococcus aureus (11 mm), and Bacillus subtilis (9 mm) at a concentration of 80 µl. For antifungal activity, the highest inhibition zone (17 mm) was observed against Penicillium chrysogenum, followed by Candida albicans (15 mm), Aspergillus niger (12 mm) and Trichoderma reesei (9 mm) at 80 µl. The synthesised TiO₂ nanoparticles successfully inhibited the bacterial cells and showed green (live) and red (dead) fluorescence in the live/dead cell viability assay. The green-synthesised titanium NPs could inhibit bacterial and fungal growth, highlighting their potential use in biomedicine and healthcare.