Tissue mimicking hydrogels can help us understand how viscoelastic properties (stiffness, elastic modulus, etc.) of biological tissues impact bubble nucleation. Adding impurities to hydrogels introduces inhomogeneities and increases their similarity to biological tissues. In this study, we evaluated the effect of stiffness and impurities on bubble nucleation in polyacrylamide (PA) hydrogels. Bubble nuclei were evaluated in 17.5%, 20%, and 22.5% v/v PA hydrogels, after which 0.25% w/v cholesterol crystals (maximum dimension = 0.6 mm) were embedded in the gels (n = 3 each). A 1.5 MHz focused ultrasound transducer was used to induce cavitation using 10-ms pulses with pressures ranging up to p+ = 89 MPa, and p− = 26 MPa and −6 dB focal dimensions of 9.4 × 1.2 mm (p−). Image analysis from high-speed photography showed bubble nucleation increases with increasing peak negative pressure and decreasing hydrogel stiffness. Adding cholesterol crystals largely decreases the acoustic cavitation threshold from p− = 19 MPa for 17.5% v/v hydrogels with no added impurities to p− = 12 MPa for the same concentration hydrogel with added cholesterol crystals. This suggests that hydrophobic cholesterol crystals weaken the gel or trap bubble nuclei, thus lowering the cavitation threshold. Future work includes investigating bubble nuclei in rat hepatocytes. [Work supported by NSF CAREER 1943937 and PSU Riess Fellowship.]