Real-time acoustic feedback control based on harmonic emissions of stimulated microbubbles may serve as a way to achieve reliable blood–brain barrier (BBB) opening with focused ultrasound in the brain. Previously, we demonstrated BBB opening was possible using sub-micron bubbles (aka nanobubbles) and produced comparable results to commercially available microbubbles (Optison, Definity, etc.). The harmonic emissions and acoustic control were observed to be more consistent using nanobubbles, which warrants further study of BBB opening using these agents. This study examined the stimulated acoustic emissions of nanobubbles at different concentrations both in vitro and in vivo and evaluated BBB opening under real-time acoustic feedback control across concentrations. Original nanobubbles (1011 bubbles/mL) have long in vitro persistence (7.3 ± 3.3 min) and circulation time in rats (approximately 10 min) under exposures in this study, and both degraded with dilutions. With all three tested dilutions (1:1, 1:10 and 1:100), successful BBB opening was reliably achieved under real-time feedback control.