Ultrasound-based engineering of carrier-free nanodrugs by supramolecular self-assembly has recently emerged as an innovative and environmentally friendly synthetic approach. By applying high-frequency sound waves (490 kHz) in aqueous solutions, the transformation of small chemotherapeutic and antibiotic drug molecules into carrier-free nanodrugs with anticancer and antimicrobial activities was recently achieved. The transformation of the antibiotic drug molecules, i.e., doxycycline, into stable nanodrugs (~130 nm) with selective anticancer activity was achieved without requiring organic solvents, chemical agents, or surfactants. The obtained nanodrug exhibited reactive oxygen species (ROS)-mediated cytotoxicity on human breast cancer (MDA-MB 231 cells) but a negligible antiproliferative effect on healthy fibroblast cells. Imaging by super-resolution microscopy (STORM) provided insights into the intracellular trafficking and endosomal escape of the nanodrugs. Overall, these findings suggest that small antibiotic drugs can be transformed into chemotherapeutic nanodrugs with high selectivity against cancer cells.