Ultrasound biomicroscopy (UBM) uses high frequencies to noninvasively yield high resolution real-time images. UBM can structurally and functionally assess development and disease models in the mouse. However, little has been reported on the thermal effects of tissue exposure to high-frequency ultrasound, and established guidelines for the safe use of clinical ultrasound use biophysical principles which have been adapted for diagnostic frequencies, and there are no published guidelines for the safe use of high frequency ultrasound. We assessed the potential of 40 MHz ultrasound to cause biological effects in mouse embryogenesis and at soft tissue-bone interfaces. UBM appears safe for mouse imaging, with thermal effects possible at tissue-bone interfaces.