Acoustic microscopy uses ultra-high frequency (UHF) ultrasound transducers over 80 MHz to perform high-resolution imaging. The pressure output of these transducers is unknown, as commercial calibrated hydrophones can measure pressure for transducers with frequencies only up to 80 MHz. This study used gas vesicle nanostructures (GVs) that collapse at 571 kPa to estimate the pressure of UHF transducers at 40, 80, 200, and 375 MHz. Agarose phantoms containing GVs were made, and a baseline ultrasound image was performed at low pressure to prevent GV collapse. Sections within the phantom were scanned at varying voltage to determine the GV collapse threshold. The pressure at full driving voltage was then calculated, assuming a linear relation between transducer voltage and pressure. The pressure calculated for the 40 MHz transducer was 2.2 ± 0.1 MPa at 21 °C. Using a hydrophone, the measured pressure was 2.1 ± 0.3 MPa, a difference of <2%, validating the method at this frequency. The pressure calculated for the other transducers was 2.0 ± 0.1 MPa (80 MHz), 1.2 ± 0.1 (200 MHz), and 1.05 ± 0.17 (375 MHz at 37 °C). This study addresses the challenge of estimating pressure output from UHF ultrasound transducers, demonstrating that the pressure output in the 40–400 MHz frequency range can be quantified.