As the microbubble's resonance frequency is size-dependent, polydisperse microbubbles induce varying drug delivery outcomes at one ultrasound frequency. This study aimed to investigate drug delivery outcome of monodisperse MBs (mMBs) with radii ranging from 1.5–2.9 μm insonified at 2 MHz. Phospholipid-coated mMBs were generated using the Horizon microfluidic flow-focusing device. In vitro experiments were conducted on single microbubble-endothelial cells (n = 68) using confocal microscopy and 10 Mfps ultra-high-speed imaging. At 220 kPa PNP for 10 cycles, the 1.5 μm mMBs exhibited the highest PI uptake in 81.3% of cases, followed by 77.3% for 2.2 μm, 36.8% for 2.7 μm and 13.3% for 2.9 μm mMBs. Conversely, the 1.5 μm mMBs had the second lowest excursion amplitude (R;max-R;0) of 0.8 ± 0.3 μm as this was 1.2 ± 0.3 μm for 2.2 μm mMBs, 1.0 ± 0.3 μm for 2.7 μm mMBs, and 0.7 ± 0.2 μm for 2.9 μm mMBs. Additionally, for the 1.5, 2.2, and 2.7 μm mMBs, PI uptake and tunnel formation occurred more often (1.6, 1.8, and 1.3 times, respectively) than PI uptake and a resealing membrane pore. During insonification, mMB pinch-off occurred more frequently in tunnel formation (70.8%) than resealing pore formation (53.3%). This research revealed the impact of mMBs size on drug delivery outcome.