We previously employed ultrasound as a needleless approach to deliver macromolecules via the transscleral route to the back of the eye in live animals (Suen et al., 2013). Here, we investigated the nature of the ultrasound-enhanced transport through sclera, the outermost barrier in the transscleral route. Thus, the possible role of cavitation from ultrasound was explored; its effect during and after sonication on scleral penetration was measured; and the dependence on the size of macromolecules was determined. We applied ultrasound frequency from 40 kHz to 3 MHz at ISATA (spatial-average-temporal-average intensity) of 0.05 W/cm2 to fresh rabbit sclera ex vivo. Fluorescent dextran of size 20 kDa to 150 kDa was used as macromolecular probes. We measured the distance of penetration of the probes through the sclera over 30 s during sonication and over 15 min after sonication from cryosectioned tissue images. Deeper penetration in the sclera was observed with decreasing frequency. The presence of stable cavitation was further verified by passive acoustic detection. The effect during sonication increased penetration distance up to 20 fold and was limited to macromolecular probes ≤ 70 kDa. The effect post sonication increased penetration distance up to 3 fold and attributed to the improved intrasscleral transport of macromolecules ≥ 70 kDa. Post-sonication enhancement diminished gradually in 3 h. As the extent of cavitation increased with decreasing frequency, the trend observed supports the contribution of (stable) cavitation to enhancing transport through sclera. Effect during sonication was attributed to flow associated with acoustic microstreaming. Effect post sonication was attributed to the temporary increase in scleral permeability. Flow-associated effect was more pronounced but only applied to smaller macromolecules.