For the first time, the three-dimensional (3D) internal structure of naturally produced Didymosphenia geminata frustules were nondestructively visualized at sub-100 nm resolution. The well-optimized hierarchical structures of these natural organisms provide insight that is needed to design novel, environmentally friendly functional materials. Diatoms, which are widely distributed in freshwater, seawater and wet soils, are well known for their intricate, siliceous cell walls called ‘frustules’. Each type of diatom has a specific morphology with various pores, ribs, minute spines, marginal ridges and elevations. In this paper, the visualization is performed using nondestructive nano X-ray computed tomography (nano-XCT). Arbitrary cross-sections through the frustules, which can be extracted from the nano-XCT 3D data set for each direction, are validated via the destructive focused ion beam (FIB) cross-sectioning of regions of interest (ROIs) and subsequent observation by scanning electron microscopy (SEM). These 3D data are essential for understanding the functionality and potential applications of diatom cells.