This book constitutes the refereed proceedings of the 16th International Workshop on Machine Learning in Medical Imaging, MLMI 2025, held in Conjunction with MICCAI 2025, Daejeon, South Korea, on September 23, 2025.The 65 full papers included in this book were carefully reviewed and selected from 101 submissions. They focus on advanced scientific research covering topics such as deep learning, foundation models, generative learning, and statistical methods with their applications to computer-aided diagnosis, multi-modality fusion, image reconstruction, digital pathology, and large-scale data analytics.