We performed a cross-sectional survey on vaccination-related knowledge, attitudes, and practices (KAP) among randomly selected parents of <5 years-old children, elderly populations (aged ≥ 55 years), and health care workers (HCWs) in 10 health zones from 4 provinces of the Democratic Republic of Congo (DRC). Questionnaires targeted both routine (BCG, measles, polio) and outbreak-related (cholera, Ebola, COVID-19) vaccinations. In total, 2751 participants were included, 1165 parents, 1040 elderly, and 546 HCWs. In general, KAP expressed were supportive of vaccination uptake, although concerns regarding side effects and feelings of being insufficiently informed were more prevalent among parents and the elderly. Vaccine acceptance was lower for outbreak vaccinations (57%) than for routine vaccinations (90%). HCWs expressed the highest vaccine acceptance. Problems with the vaccine supply chain were reported by 20% of respondents. Despite a high level of positive KAP towards vaccination, parents and the elderly expressed a need to be better informed and had concerns regarding vaccine side-effects. A high acceptance for routine vaccinations was reported by participants, but somewhat less for outbreak vaccinations. In conclusion, HCWs in the communities could play a key role in the increased uptake of routine vaccinations and in optimizing uptake during outbreaks, provided that the supply chain is functioning well.