Speaking up by healthcare professionals (HCPs) is an important resource to reduce risks to patient safety. Due to complex tradeoffs, HCPs are often reluctant to voice their concerns. A survey investigated HCPs' likelihood to speak up. A cross-sectional survey study among HCPs in 5 Swiss hospitals addressed speaking-up behaviors, safety climate, and likelihood to speak up about poor hand hygiene practice described in a vignette. Likelihood to speak up was analyzed using a multilevel regression model. Of surveyed HCPs (n = 1217), 56% reported that they would speak up to a colleague with poor hand hygiene practice. Nurses as compared to doctors rated the situation as more realistic (5.25 vs 4.32, P < .001), felt more discomfort with speaking up (4.00 vs 3.34, P < .001), and reported a slightly lower likelihood of speaking up (4.41 vs 4.77, P < .001). Clinical function (hierarchy) was strongly associated with speaking-up behavior (P < .001). Higher risk of harm to the patient (P < .001) and higher frequencies of past speaking-up behaviors (P = .006) were positively associated with the likelihood to speak up. Higher frequencies of past withholding voice (P = .013) and higher levels of resignation (P = .008) were both associated with a lower likelihood to speak up. Infection control interventions should empower HCPs to speak up about non-adherence with prevention practices by addressing authority gradients and risk perceptions and by focusing on resignation.