Sonochemical synthesis, driven by acoustic cavitation, has emerged over the past two decades as a powerful and versatile method for producing nanoparticles of bioactive substances with enhanced physicochemical and biological properties. The extreme conditions generated by the collapse of cavitation bubbles – such as transient high temperatures and pressures – facilitate molecular fragmentation, nucleation, and controlled nanoparticle growth. These characteristics make sonochemistry particularly well suited for nanosizing pharmaceuticals, enzymes, and natural bioactive compounds. In addition to a wide range of experimental techniques, recent advances in molecular dynamics simulations have offered critical insights into the molecular-level mechanisms underlying sonochemical processes, revealing the roles of solvent interactions and interfacial dynamics in determining nanoparticle size and stability. This deeper understanding has enabled more precise tuning of synthesis parameters to achieve desired nanoparticle characteristics. Sonochemically synthesized nanoparticles have demonstrated significant potential in diverse biomedical fields, including targeted drug delivery, sonodynamic therapy, regenerative medicine, and antimicrobial coatings. This minireview consolidates two decades of research, highlighting key advancements in the sonochemical synthesis of nanoparticles of bioactive substances, with a focus on molecular-level insights, biomedical applications, future research directions, ongoing challenges, and future perspectives.