The antimicrobial drug resistance is increasing with the passage of time due to wide and improper use of broad spectrum drugs and the demand of the new drug increases day by day. The present study was planned to encounter this problem by synthesizing titanium dioxide nanoparticles (TiO2 NPs) by an eco‐friendly route using Cannabis sativa leaves extract. The synthesized TiO2 NPs were calcined at 100, 300, 600, and 900°C in a muffle furnace. The crystallographic parameters were studied by X‐ray diffraction and the phase transition occurred above 600°C. The surface morphology of the synthesized samples was studied by transmission electron microscopy (TEM), and scanning electron microscopy (SEM) and the particle size was measured through the ImageJ software. The elemental composition and purity of all the samples were studied by performing energy dispersive X‐ray (EDX). All the synthesized TiO2 NPs were tested for their antimicrobial effect against Gram‐positive and Gram‐negative bacteria using the agar well diffusion method. The activity was found higher against Gram‐negative bacteria and compared to Gram‐positive bacteria.