Quantitative assessment of micro-structure of materials is of key importance in many fields including tissue engineering, biology, and dentistry. Micro-computed tomography (µ-CT) is an intensively used non-destructive technique. However, the acquisition parameters such as pixel size and rotation step may have significant effects on the obtained results. In this study, a set of tissue engineering scaffolds including examples of natural and synthetic polymers, and ceramics were analyzed. We comprehensively compared the quantitative results of µ-CT characterization using 15 acquisition scenarios that differ in the combination of the pixel size and rotation step. The results showed that the acquisition parameters could statistically significantly affect the quantified mean porosity, mean pore size, and mean wall thickness of the scaffolds. The effects are also practically important since the differences can be as high as 24% regarding the mean porosity in average, and 19.5 hours and 166 GB regarding the characterization time and data storage per sample with a relatively small volume. This study showed in a quantitative manner the effects of such a wide range of acquisition scenarios on the final data, as well as the characterization time and data storage per sample. Herein, a clear picture of the effects of the pixel size and rotation step on the results is provided which can notably be useful to refine the practice of µ-CT characterization of scaffolds and economize the related resources.