Pharmaceutical drug therapy is often hindered by issues caused by poor drug selectivity, including unwanted side effects and drug resistance. Spatial and temporal control over drug activation in response to stimuli is a promising strategy to attenuate and circumvent these problems. Here we use ultrasound to activate drugs from inactive macromolecules or nano-assemblies through the controlled scission of mechanochemically labile covalent bonds and weak non-covalent bonds. We show that a polymer with a disulfide motif at the centre of the main chain releases an alkaloid-based anticancer drug from its β-carbonate linker by a force-induced intramolecular 5- exo - trig cyclization. Second, aminoglycoside antibiotics complexed by a multi-aptamer RNA structure are activated by the mechanochemical opening and scission of the nucleic acid backbone. Lastly, nanoparticle–polymer and nanoparticle–nanoparticle assemblies held together by hydrogen bonds between the peptide antibiotic vancomycin and its complementary peptide target are activated by force-induced scission of hydrogen bonds. This work demonstrates the potential of ultrasound to activate mechanoresponsive prodrug systems.