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Advances in microfabrication technology, and its increasing accessibility, allow us to explore fungal biology as never before. By coupling molecular genetics with fluorescence live-cell imaging in custom-designed chambers, we can now probe single yeast cell responses to changing conditions over a lifetime, characterise population heterogeneity and investigate its underlying causes. By growing filamentous fungi in complex physical environments, we can identify cross-species commonalities, reveal species-specific growth responses and examine physiological differences relevant to diverse fungal lifestyles. As affordability and expertise broadens, microfluidic platforms will become a standard technique for examining the role of fungi in cross-kingdom interactions, ranging from rhizosphere to microbiome to interconnected human organ systems. This review brings together the perspectives already gained from studying fungal biology in microfabricated systems and outlines their potential in understanding the role of fungi in the environment, health and disease., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)