Investigations have been carried out with the goal of assessing the trabecular bone thickness of biological samples using images obtained by micro-computed tomography and magnetic resonance imaging. There is no conventional definition of trabecular bone thickness, and many methods may be involved in determining it. However, the results of the available algorithms or software packages differ considerably from each other. This paper determines trabecular bone thickness on the basis of several algorithms. A deep understanding of the performance of different methods is achieved by studying pseudo-three-dimensional images of both geometrical models of well-defined thickness and real bone samples with different bone densities. The models facilitate comparisons between the algorithms or software packages. Comparison of the results obtained from these commercial software packages and other state-of-the-art algorithms shows that the thickness, spatial distribution, and shape of an object affect each result differently, but in a significant manner. This is primarily due to variations in the thresholding algorithms used to distinguish object area elements (pixels/voxels) from the background, or non-object, region. Additionally, the results show that the average difference in thickness measurements can vary by up to 102.34% for models and 46.49% for real bone samples. This data shows that the differences in measurements of the trabecular bone thickness due simply to the algorithm involved are remarkable. Therefore, biomedical engineers and scientists should be careful to select the algorithm that is most compatible with their specific application.