Nanobiotechnology has offered new approaches for drug delivery and for the development of medicines against microbial infections including the deadly multidrug-resistant tuberculosis (MDR-TB). The present study deals with the antimycobacterial activity of titanium dioxide (TiO₂) nanoparticles against Mycobacterium tuberculosis. Initially, TiO₂ nanoparticles were synthesized from titanium(IV)oxysulfate (TiOSO₄) solution using the sol–gel method. The shape and size of the TiO₂ crystals were analyzed using TEM and SEM and were found to be spherical and 16 nm respectively, thereby indicating their morphology. Moreover, the XRD pattern confirms that the prepared TiO₂ nanoparticles were anatase as well as being tetragonal and FTIR spectra show the stretching vibrations of Ti–O and Ti–O–Ti. Further, the TiO₂ nanoparticles were shown to exhibit excellent size-dependent antimycobacterial activity against Mycobacterium tuberculosis, M. bovis and Mycobacterium sp. in a concentration dependent manner (10–100 μg mL⁻¹). Confocal laser scanning microscopic analysis also confirmed that the TiO₂ nanoparticles inhibit the biofilm formation of all three tested mycobacteria. The metabolic activity of mycobacteria was decreased up to 3–4 fold with an increase in the concentration of the TiO₂ nanoparticles hence affecting the biofilm formation. Moreover, the TiO₂ nanoparticles were less toxic (>25%) against normal lung bronchus cells at 100 μg mL⁻¹ after 24 h treatment revealing that the nanoparticles are less toxic in nature. Taken together, these results showed that the TiO₂ nanoparticles can be a potential biofilm agent against mycobacteria.