Hydrostatic pressure (HP) and osmotic stress (OS) play an important role in various biological processes, such as cell proliferation and differentiation. In contrast to canonical mechanical signals transmitted through the anchoring points of the cells with the extracellular matrix, the physical and molecular mechanisms that transduce HP and OS into cellular functions remain elusive. Three-dimensional cell cultures show great promise to replicate physiologically relevant signals in well-defined host bioreactors with the goal of shedding light on hidden aspects of the mechanobiology of HP and OS. This review starts by introducing prevalent mechanisms in the generation of HP and OS signals in biological tissues that are subject to pathophysiological mechanical loading. We then revisit various mechanisms on the mechanotransduction of HP and OS, and describe the current state of the art in bioreactors and biomaterials for the control of the corresponding physical signals.