Ultrasound and microbubble optimization studies for therapeutic applications are often conducted in water/saline, with a fluid viscosity of 1 cP. In an in vivo context, microbubbles are situated in blood, a more viscous fluid (∼4 cP). In this study, ultrahigh-speed microscopy and passive cavitation approaches were employed to investigate the effect of fluid viscosity on microbubble behavior at 1 MHz subject to high pressures (0.25-2 MPa). The propensity for individual microbubble (n = 220) fragmentation was found to significantly decrease in 4-cP fluid compared with 1-cP fluid, despite achieving similar maximum radial excursions. Microbubble populations diluted in 4-cP fluid exhibited decreased wideband emissions (up to 10.2 times), and increasingly distinct harmonic emission peaks (e.g., ultraharmonic) with increasing pressure, compared with those in 1-cP fluid. These results suggest that in vitro studies should consider an evaluation using physiologic viscosity perfusate before transitioning to in vivo evaluations.