Abstract Pre-existing health and economic challenges mean residents of low- and middle-income countries (LMICs) are likely to be particularly vulnerable to infectious disease pandemics. Limited access to hygiene facilities, water, soap and masks, and dense living environments impeded effective practice of preventive behaviours – handwashing with soap (HWWS), mask wearing and physical distancing – a key line of primary defence against COVID-19. Here we describe a multi-country analysis of prevalence of key hygiene prevention behaviours and their determinants associated with an international non-governmental organisation (WaterAid) hygiene behaviour change programmes for COVID-19 prevention. The goal of this analysis is to inform future outbreak preparedness and pandemic response in LMICs. Cross-sectional household surveys were conducted in October-November 2020 in seven countries where WaterAid worked (Ethiopia, Ghana, Nepal, Nigeria, Rwanda, Tanzania and Zambia). Multivariable mixed-effects regression analyses were used to explore relationships between self-reported behavioural outcomes of interest (handwashing with soap, physical distancing, and mask use) and demographic characteristics, behavioural factors (knowledge, norms, barriers, motives), and exposure to COVID-19 communications. Most respondents (80%) reported increasing their handwashing behaviour after the pandemic, but practice of HWWS at COVID-19-specific prevention moments was low. Mask wearing (58%) and physical distancing (29%) varied substantially between countries. Determinants of key behaviours were identified, including age and socioeconomic status, perceived norms, self-regulation, and the motive of protecting others. These findings highlight that leveraging behaviour-specific emotional drivers and norms, reducing common barriers and promoting targeted messages about specific behaviours and actions individuals can take to reduce risk are necessary to support large-scale behaviour change. Learning from the COVID-19 response to more effectively integrate novel behaviours into existing health promotion will be vital for disease prevention and outbreak resilience.Key messages What is already known on this topic Facilitating COVID-19 prevention behaviours of hand hygiene, mask use and physical distancing in low- and-middle income countries comes with unique challengesIdentifying effective strategies to promote adoption of key behaviours in diverse contexts over a period of rapid change will be key for future pandemic preparednessWhat this study adds This multi-country analysis of areas where WaterAid implemented an initial mass media COVID-19 response in 2020 observed lower practice of handwashing at novel COVID-19 prevention moments compared to established moments and variable physical distancing behaviour, and examined behaviour-specific determinants and normsHow this study might affect research, practice or policy Renewed focus on identified key drivers of behaviour: targeting critical age-groups and vulnerable populations, increasing descriptive norms and motives of protecting others and respect, and reducing common barriers, with targeted messaging for novel handwashing moments, may be key to ongoing COVID-19 responseLearning from the rapid COVID-19 response on how well we are able to promote novel behaviours alongside established ones in a variety of contexts can inform future disease prevention and outbreak resilience.