The aim of this article is to verify the applicability of impulse acoustic microscopy as a tool for evaluating scaffolds in tissue engineering. Decellularized rat diaphragms and nonwoven polymer scaffolds made from cellulose diacetate, chlorinated polyvinyl chloride, and polysulfone were used as model objects. Optical microscopy, scanning electron microscopy, and histological research were used as reference methods in order to realize feasibility of the acoustic microscopy method in a regenerative medicine field. Direct comparison of different methods was carried out. The high frequency ultrasound microscopy is defined as a useful non-invasive tool for studying and investigating the bulk morphology and bulk structure of opaque scaffolds, which allows to diagnose and to assume heterogeneous regions and defects inside the specimens. POLYM. ENG. SCI., 57:709-715, 2017. (c) 2017 Society of Plastics Engineers