<p>The nucleolus is the site of ribosomal RNA synthesis and ribosome biogenesis. Advances in high-resolution imaging and next-generation sequencing have unveiled unprecedented details of its intricate architecture and dynamic organization. In this Review, we focus on the dynamic organization of mammalian nucleoli, while drawing selective comparisons with other organisms to highlight conserved and divergent principles of nucleolar organization. We discuss recent progress in deciphering the multilayered compartments of nucleoli, the physical principles driving their organization and dynamics, and their functional interplay during stepwise ribosomal RNA processing, ribosome assembly and maintenance of compartment integrity. We also discuss how disruptions of nucleolar structure–function relationships can drive cellular stresses and diseases, offering new opportunities for therapeutic interventions.</p>