Highlights  Using tapping mode imaging, the thickness of microbubble shells can be quantified  Quantifying shell thickness has significant impact on Young's Modulus values  Atomic force microscopy can be used to quantify thickness of the PEG brush layer ABSTRACT: Microbubbles (MBs), which are used as ultrasonic contrast agents, have distinct acoustic signatures which enable them to significantly enhance visualisation of the vasculature. Research is progressing to develop MBs which act as drug/gene delivery vehicles for site-specific therapeutics. In order to manufacture effective theranostic vehicles, it is imperative to understand the mechanical and nanostructural properties of these agents; this will enrich the understanding of how the structural, biophysical and chemical properties of these bubbles impact their functionality. We produced microfluidic phospholipid-based MBs due to their favourable properties, such as biocompatibility and echogenicity, as well as the ability to modify the shell for targeting applications. We have drawn upon atomic force microscopy to conduct force-spectroscopy and tapping-mode imaging investigations. We have, for the first time to our knowledge, been able to accurately quantify the thickness and lipid configuration of phospholipid-shelled MBs-showing a trilayer as opposed to the conventional monolayer structure. Furthermore, we have measured MB stiffness and employed different mechanical theories to quantify the Young s Modulus. We show that the Reissner theory is inappropriate for mechanical characterisation of phospholipid MBs, however, the Hertz model does offer biologically relevant comparisons. Analysis using the Alexander-de Gennes polymer brush theory has allowed us to provide new information regarding how the thickness of the polyethylene glycol brushes, end-grafted to our phospholipid microbubbles, changes with diameter.