In the present work, the effect of pure and different weight % of Cobalt (Co) doped titanium dioxide (TiO2) nanoparticles (NPs) were developed from sol–gel approach. The powder x-ray diffraction pattern of pure and 1%, 3%, and 5% Co-doped TiO2 NPs confirms the anatase phase structure and average crystallite size was obtained from 37, 38, 42 and 48 nm respectively for pure TiO2 and 1%, 3%, and 5% Co-doped TiO2. The spherical-like morphology was observed from the prepared materials which were primarily confirmed by the FESEM analysis. The optical behaviour of the developed material was investigated by UV–vis spectrum and energy band gab was achieved to be 1.95 eV, 3.96 eV, 3.89 eV and 3.62 eV respectively for pure TiO2, 1 wt%, 3 wt% and 5 wt% Co-doped TiO2 NP. The Kirby Bauer Agar Well Diffusion Assay method was employed to explore antimicrobial activity of nanosized pure and Co-doped TiO2 colloidal suspension against the test microorganisms two Gram positive bacteria (Staphylococcus aureus, Bacillus subtilis), two Gram negative Bacteria (Escherichia coli, Pseudomonas aeruginosa), and two fungi (Candida albicans, Aspergillus niger). It shows that the Co-doped TiO2 NPs inhibited the multiplication and growth of the above mentioned test bacteria and fungi. Antimicrobial activity was found against all tested microorganisms and confirmed that Co-doped TiO2 NPs possess higher antimicrobial activity at various concentration range of 25 μg, 50 μg, 75 μg and100 μg respectively for 1 wt%, 3 wt% and 5 wt% Co-doped TiO2 solutions.