The use of nanobiotechnology for the development of nanomaterials using environmentally friendly procedures and having combined ability to fight efficiently antibiotic-resistant biofilm-forming bacteria and having low toxicity remains challenging is critical for environmental and biomedical applications. In the present study, the experimental data obtained by coupled gas chromatography-mass spectrometry, Fourier transform infrared spectroscopy (FTIR), and high-performance liquid chromatography suggested that the polysaccharide rich algal extract simultaneously acted as bio-reduction and capping agent for the successful formation of TiO2 nanoparticles (NPs) that showed anti-oxidant properties. The results obtained by ultraviolet-visible diffuse reflectance spectroscopy and FTIR indicated the presence of algal functional groups onto the surface of the TiO2 NPs. Powder XRD analysis confirmed the polycrystalline nature of the TiO2 NPs having tetragonal crystal structure. Field emission scanning and transmission electron microscopy as well as energy dispersive X-ray spectroscopy and elemental mapping revealed the formation of agglomerated non-spherical structures. The obtained TiO2 NPs inhibited the formation of biofilms and stopped the metabolic respiration of A. baumannii and K. pneumoniae at 250 mu g/mL. Anti-virulence and anti-pathogenicity effect of TiO2 NPs due to the excessive production of reactive oxygen species on bacterial membrane was confirmed by confocal laser scanning microscopy. Scanning electron microscopy observation of cell integrity loss, architectural damage, and extracellular damage revealed that the obtained biosynthesized TiO2 NPs had enhanced bio-efficiency against the tested pathogens. Lastly, a reduced death rate of A. franciscana was observed, suggesting that the biosynthesized TiO2 NPs had low toxicity against that model. As a result, the present work demonstrated the potential of safe and biological methods for the production of TiO2 NPs, improving their biological features against A. baumannii and K. pneumoniae and indicating their potential for clinical use.