The occurrence of cavitation in the brain from blunt impact is difficult to investigate and remains controversial. The objective of this study was to investigate cavitation formation from blunt impact, without compromising the cranial cavity, using novel acoustic biomarkers. These biomarkers were developed using a laser-induced cavitation study, acrylic surrogate impact tests, and fresh non-frozen pig cadaver impact tests. Laser-induced cavitation displayed a ~125 and 250 kHz response on high frequency sensors, and a ~13–20 kHz, ~50–70 kHz and ~100–140 kHz frequency response on hydrophones. For surrogate tests, a harmonic response (~50–70, 100–140 and 250 kHz) was recorded 0–5 ms following confirmed cavitation impacts. In cadaver tests, largest impacts elicited a coupled low frequency (~50–70 kHz) and high frequency (~100–140 kHz) acoustic response 0–5 ms following impact. Negative control pig cadaver head tests provided low frequency response but were not associated with high frequency content for any impact. Using a series of validation tests and negative controls, this study developed an acoustic biomarker, a low frequency response coupled with a high frequency harmonic, which is sensitive and specific to CSF cavitation without compromising the cranial cavity. This provided evidence of cavitation within a fresh pig cadaver skull upon blunt impact.