The ability to use sound or ultrasound (US) to modify plant growth in vitro, and if possible, to improve yield or productivity, would benefit horticultural scientists. In this study, potato (Solanum tuberosum L. cv. Desirée) in vitro node segments with a single leaf were exposed to US (35 kHz, 70 W, for 20 min). Morphological, physiological and biochemical parameters were measured. Treatment with US 24 h after ultrasonication temporarily accelerated shoot growth but inhibited the development and growth of roots due to a decrease in the level of AA directly after ultrasonication. At the end of the subculture period, i.e., 4 weeks after US treatment, shoot length increased 20% more than control shoots after 4 weeks, while shoot fresh weight was 24% higher than that of control shoots, representing the long-term after-effect of the US treatment. The antioxidant defence system was induced, partly by intensive plantlet growth and development from node segments, and partly by abiotic stress caused by the US treatment. Immediately (0 h) or 24 h after ultrasonication, superoxide dismutase, ascorbate peroxidase, and glutathione reductase activity increased significantly, as did the concentration of low molecular weight antioxidants (GSSG, GSH, AA, TCPa). However, there was no glutathione peroxidase activity, most likely due to the lack of selenium in the basal in vitro growth medium. Therefore, the glutathione-S-transferase path of the ascorbate-glutathione pathway was induced both by metabolic processes and by abiotic stresses and took part in the reduction of organic peroxides using glutathione. US treatment ameliorated the ratios of ascorbic acid/glutathione and reduced/oxidized glutathione, ensuring the development of plantlets with significantly improved shoot parameters, such as higher shoot length and fresh weight, by the end of the subculture period.