Label-free optical imaging employs natural and non-destructive approaches to visualize biomedical samples for both biological assays and clinical diagnosis. At present, this field revolves around multiple technology-oriented communities, each with a specific focus on a particular modality, despite the existence of shared challenges and applications. As a result, biologists or clinical researchers who require label-free imaging are often not aware of the most appropriate modality to use. This Review presents a comprehensive overview of, and comparison among, different label-free imaging modalities and discusses common challenges and applications. We expect this Review to facilitate collaborative interactions between imaging communities, push the field forwards and foster technological advancements and biophysical discoveries, as well as facilitate new avenues in clinical detection, diagnosis and monitoring of diseases.