We present a novel class of ultrasound (US) contrast agents, which enable de novo production of echogenic O2 microbubbles (MBs) in vivo in regions with elevated hydrogen peroxide (H2O2). The agent is a 200 nm silica shell nanoparticle loaded with catalase (catSHEL) that catalyzes H2O2 into water (H2O) and oxygen (O2). Importantly, this reactive process produces O2 MBs visible during US imaging. We have shown that this agent can detect elevated levels of H2O2 in kidneys with acute kidney injury (AKI). The purpose of this in vitro study is to further characterize the US properties of these O2 MBs. When exposed to US energy (mechanical index, MI = 0.07), a marked increase in the 2nd harmonic signal was recorded after the addition of H2O2 when the catSHELs were suspended in 25% plasma + phosphate buffered saline (PBS) versus PBS alone (13.1 vs. 1.7 dB). This harmonic gain abated at higher plasma concentrations (6.0 dB at 100% plasma) and higher (MI = 0.10) and lower (MI = 0.03) US transmit powers (8.8 and 5.7 dB, respectively). The harmonic signal also increased with increasing surfactant concentration. Again, the response was greatest at moderate acoustic power. O2 MBs produced in PBS alone were transient and relatively large, while those produced at increasing plasma and surfactant concentrations were smaller and longer lasting, suggesting that they are stabilized in plasma or surfactant. Overall, nonlinear US imaging may be suitable for the local visualization of O2 MBs produced in response to AKI.