The sensitivity of the acoustic response of microbubbles, specifically a strong correlation between their subharmonic response and the ambient pressure, has motivated development of a non-invasive subharmonic-aided pressure estimation (SHAPE) method. However, this correlation has previously been found to vary depending on the microbubble type, the acoustic excitation and the hydrostatic pressure range. In this study, the ambient pressure sensitivity of microbubble response was investigated. The fundamental, subharmonic, second harmonic and ultraharmonic responses from an in-house lipid-coated microbubble were measured for excitations with peak negative pressures (PNPs) of 50-700 kPa and frequencies of 2, 3 and 4 MHz in the ambient overpressure range 0-25 kPa (0-187 mmHg) in an in vitro setup. The subharmonic response typically has three stages-occurrence, growth and saturation-with increasing excitation PNP. We find distinct decreasing and increasing variations of the subharmonic signal with overpressure that are closely related to the threshold of subharmonic generation in a lipid-shelled microbubble. Above the excitation threshold, that is, in the growth-saturation phase, subharmonic signals decreased linearly with slopes as high as -0.56 dB/kPa with ambient pressure increase; below the threshold excitation (at atmospheric pressure), increasing overpressure triggers subharmonic generation, indicating a lowering of subharmonic threshold, and therefore leads to an increase in subharmonic with overpressure, the maximum enhancement being ∼11 dB for 15 kPa overpressure at 2 MHz and 100 kPa PNP. This study indicates the possible development of novel and improved SHAPE methodologies.