The goals of this study were to observe the spread of viruses in a hotel setting and to assess the effectiveness of a hygiene intervention in reducing their spread. Selected fomites in one hotel room were inoculated with bacteriophage ϕx-174, and fomites in a conference center within the same hotel were inoculated using bacteriophage MS2. Cleaning of the contaminated room resulted in the spread of viruses to other rooms by the housekeeping staff. Furthermore, viruses were transferred by hotel guests to the conference center and a communal kitchen area. Additionally, conference attendees transferred viruses from the conference center to their hotel rooms and a communal kitchen area. This study demonstrated how viruses can be spread throughout a hotel setting by both housekeepers and guests. A hygiene intervention, which included providing hand hygiene products and facial tissues to the guests and disinfecting solutions with disposable wipes to the housekeeping staff, was successful in reducing the spread of viruses between the hotel guest rooms and conference center. The hygiene intervention resulted in significantly reduced transfer of the ϕx-174 between the contaminated hotel room and other hotel rooms, communal areas, and the conference center (p = 0.02).