Background Hand hygiene compliance is considered the most (cost-)effective measure for preventing health care–associated infections. While hand hygiene interventions have frequently been implemented and assessed in hospitals, there is limited knowledge about hand hygiene compliance in other health care settings and which interventions and implementation methods are effective. Objective This study aims to evaluate the effect of a multimodal intervention to increase hand hygiene compliance of nurses in nursing homes through a cluster randomized controlled trial (HANDSOME study). Methods Nursing homes were randomly allocated to 1 of 3 trial arms: receiving the intervention at a predetermined date, receiving the identical intervention after an infectious disease outbreak, or serving as a control arm. Hand hygiene was evaluated in nursing homes by direct observation at 4 timepoints. We documented compliance with the World Health Organization’s 5 moments of hand hygiene, specifically before touching a patient, before a clean/aseptic procedure, after body fluid exposure risk, after touching a patient, and after touching patient surroundings. The primary outcome is hand hygiene compliance of the nurses to the standards of the World Health Organization. The secondary outcome is infectious disease incidence among residents. Infectious disease incidence was documented by a staff member at each nursing home unit. Outcomes will be compared with the presence of norovirus, rhinovirus, and Escherichia coli on surfaces in the nursing homes, as measured using quantitative polymerase chain reaction. Results The study was funded in September 2015. Data collection started in October 2016 and was completed in October 2017. Data analysis will be completed in 2020. Conclusions HANDSOME studies the effectiveness of a hand hygiene intervention specifically for the nursing home environment. Nurses were taught the World Health Organization’s 5 moments of hand hygiene guidelines using the slogan “Room In, Room Out, Before Clean, After Dirty,” which was developed for nursing staff to better understand and remember the hygiene guidelines. HANDSOME should contribute to improved hand hygiene practice and a reduction in infectious disease rates and related mortality. Trial Registration Netherlands Trial Register (NTR6188) NL6049; https://www.trialregister.nl/trial/6049 International Registered Report Identifier (IRRID) DERR1-10.2196/17419