Environmental sanitation is crucial especially in organizations such as hotels that cater to medical tourism, daycare facilities, and nursing homes. This is because the majority of the population may be immunocompromised. The goal of this study was to investigate the efficacy of Titanium Dioxide Nanoparticles (TDN) against environmental microorganisms found on hotel room fomites (e.g. remote control, door knobs, and bathroom floors) before and after treatment with TDN with hotel room blinds open (since TDN is a photocatalyticcompund). Following this, the effect of TDN was studied on coupons (porous=carpet and non-porous=Formica) inoculated with specific pathogenic bacteria (Salmonella, Listeria, and E. coli O157:H7) and viral surrogates (MS2). These studies were carried out under dark and light conditions. The results showed a reduction in Salmonella (98.03% and 22%), E. coli (97.77% and 97.8%) and MS2 (23% and 15.7%) under both light and dark conditions respectively; although, increased reduction in bacterial CFU and viral PFU were observed under light conditions Reduction of Listeria was not observed under either dark or light conditions. This could be because of difference in membrane composition between Gram-positive and Gram-negative microorganisms. The results showed that the use of TDN for environmental sanitation is effective and could reduce the risk of infections due pathogenic microorganisms transfer via fomites.