The production of  TiO2 NPs using Staphylococcus aureus was simplified and made more efficient. Titanium sulfate concentration, reaction time, temperature, and pH were among the variables optimized. The ideal conditions were a titanium sulfate concentration of 0.025M, a reaction period of 30 minutes, a temperature of 60°C, and a pH of 7. Bacteria isolated from otitis media infections were shown to be susceptible to the in vitro activity of TiO2 NPs. AFM, SEM, and FTIR spectroscopy were among the tools and techniques used to characterize the produced nanoparticles. The TiO2 NPs measured 40 to 50 nm in size and had a spherical shape. Based on the results of the antibiotic susceptibility test, it was found that the bacterial isolates tested were sensitive, resistant, or moderately resistant to four different antibiotics. To test the sensitivity of several G⁺ and G^_ bacteria to 5 different doses of TiO2 nanoparticles, 25, 50, 100, 200, and 400 μg/ml were used. These bacteria included multidrug-resistant Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiella pneumoniae. The pathogenic bacteria Staphylococcus aureus and Streptococcus pneumoniae necessitated a concentration of 100 μg/ml of TiO2 nanoparticles to inhibit their growth, unlike Klebsiella pneumoniae.