Biofilms are essential to the aquatic environment. Recent advances in technology resulted in increased use of nanomaterials (such as titanium dioxide nanoparticles) and their release into aquatic environments with unknown long-term effects. Potential toxicity of titanium dioxide, known for its photocatalytic properties, on Pseudomonas aeruginosa (PAO1-gfp) and Pseudomonas sp. (CT07-gfp) biofilm formation and proliferation was assessed using flowcells, confocal laser scanning microscopy (CLSM), and total and viable cell release into effluent under different titanium dioxide concentrations (100 ppm, 10 ppm and 1 ppm). COMSTAT software analysis was used to obtain quantitative morphological biofilm data. Results showed that titanium dioxide had a concentration and media-dependent effect on biofilm formation, growth, proliferation and viability. Viable effluent cell counts remained within the same order of magnitude. Biofilm recovery was evident within 24-48 hours after exposure. At environmentally relevant concentration (1 ppm), there was no effect on formation, proliferation or growth of the biofilm.