This Special Issue focuses on the effects of hydrostatic pressure on biological systems and the use of these effects for exploring the structure, function, and molecular dynamics of biological macromolecules and their ensembles. Here, we present a selection of papers highlighting new experimental findings and new theoretical concepts in high-pressure biosciences. In these studies, the authors combine pressure perturbation approaches with NMR and optical spectroscopy, kinetic and thermodynamic techniques, functional genomics and transcriptomics, and molecular dynamics simulations to gain new insights into the conformational dynamics of proteins and nucleic acids and to better understand the mechanisms of high-pressure adaptation in piezophiles. The articles collected in this issue demonstrate the unique exploratory potential of the pressure perturbation approach for biochemistry, biophysics, mechanistic enzymology, and evolutionary biology.