<p>Many prokaryotic cells are encased in a para-crystalline sheath composed of lattice-forming proteins, collectively known as the surface layer (S-layer). S-layer proteins are among the most abundant proteins in archaea and bacteria. They exhibit remarkable sequence and structural diversity, while performing essential structural, protective and physiological functions. Recent advances in structural biology, cell biology and bioinformatics have reshaped our understanding of S-layer biogenesis and organization, while also revealing that S-layers are far more widespread among prokaryotes than previously envisaged. In addition, it has become increasingly clear that S-layers have crucial roles in microbial interactions and community dynamics. In this Review, we explore the architectural principles and self-assembly mechanisms that govern S-layers and examine their diverse functional roles in mediating interactions with the external environment of prokaryotes. We argue that deeper insights into these abundant surface structures are critical for understanding how they mediate multicellular interactions; a key step towards deciphering the organization of biofilms and microbiomes, which are fundamental modes of microbial life on Earth.</p>