Background Provision of donor human milk is handled by established human milk banks that implement all required measures to ensure its safety and quality. Detailed human milk banking guidelines on a European level are currently lacking, while the information available on the actual practices followed by the European human milk banks, remains limited. The aim of this study was to collect detailed data on the actual milk banking practices across Europe with particular emphasis on the practices affecting the safety and quality of donor human milk. Materials and methods A web-based questionnaire was developed by the European Milk Bank Association (EMBA) Survey Group, for distribution to the European human milk banks. The questionnaire included 35 questions covering every step from donor recruitment to provision of donor human milk to each recipient. To assess the variation in practices, all responses were then analyzed for each country individually and for all human milk banks together. Results A total of 123 human milk banks completed the questionnaire, representing 85% of the European countries that have a milk bank. Both inter- and intra-country variation was documented for most milk banking practices. The highest variability was observed in pasteurization practices, storage and milk screening, both pre- and post-pasteurization. Conclusion We show that there is a wide variability in milk banking practices across Europe, including practices that could further improve the efficacy of donor human milk banking. The findings of this study could serve as a tool for a global discussion on the efficacy and development of additional evidence-based guidelines that could further improve those practices.