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As vaccination acceptance is dynamic, the current COVID-19 pandemic may have changed maternal perceptions towards childhood vaccination and influenced their decision to vaccinate their children. This study aimed to examine the associations between socio-demographic characteristics and maternal attitudes towards childhood vaccination at the beginning of the COVID-19 pandemic in Cyprus. A web-based cross-sectional study was conducted between April and June 2020 using a self-administered survey. Adult mothers with at least one child under the age of 18 were included in this study. Overall, 703 mothers were included, with the majority of them strongly agreeing or agreeing that all vaccinations included in the National Vaccination Program should be available to children (89.3%), recognizing the usefulness of vaccines (91.4%) and their ability to provide protection (90%), acknowledging that the benefits of vaccination outweigh potential risks (86.6%), and being aware that some vaccines may cause adverse reactions (84.1%). Socio-demographic factors, particularly marital status, single-parent status, and employment status, have a significant impact on maternal attitudes and perceptions regarding childhood vaccination. Public health policies should be designed to overcome structural and healthcare system barriers specifically related to socio-demographic factors, ensuring effective and comprehensive strategies to improve childhood vaccination uptake.