The application of therapeutic ultrasound (TUS) in combination with contrast agents (USCA) to mediate gene delivery relies on the understanding of the bioeffects involved. The objective of this study was to evaluate the various bioeffects generated by albumin-coated microbubbles: Optison, an USCA, when applied with TUS operated for 10–30min, on cells and on DNA transfection. This study reveals that Optison microbubbles were still acoustically active after long-term TUS application of 30min. Optison enhances TUS-gene transfection by increasing the number of plasmids in the cells and also by distributing the plasmids to more cells, without significant decrease in cell viability. Optison also interacts with the DNA to further enhance transfection in a mechanism not necessarily involving cavitation. However, Optison affects mainly the cell cytoplasmatic membrane, without interfering with DNA intracellular trafficking. Using high-resolution scanning electron microscopy (HRSEM), the bioeffects on cell membrane induced by TUS–Optison were observed, demonstrating that Optison lead to a rougher surface, characterized by depressions that are reversible within 24-h post TUS. These effects are different from those observed when only TUS was applied. The findings from this study suggest that albumin-coated microbubbles enhances transfection when using TUS for 10–30min, and that microbubbles play a major role in elevating cell transfection level and efficiency.