Coupled dual-beam focused ion beam electron microscopy (FIB-EM) has gained popularity across multiple disciplines over the past decade. Widely utilized as a stand-alone instrument for micromachining and metal- or insulator-deposition in numerous industries, the sub-μm-scale ion milling and integrated electron imaging capabilities of such FIB-based systems are well documented in the materials science literature. These capacities make FIB-EM a powerful tool for in-situ site-specific preparation of ultrathin foils for transmission electron microscopy. Recent advancements in the field-emission guns of FIB-EM systems have provided spatial resolution comparable to that of many high-grade scanning electron microscopes, providing enhanced imaging capacities with material-deposition and material-removal capabilities. Recently, FIB-EM preparation techniques have been applied to geological samples to characterize mineral inclusions, grain boundaries, and microfossils. We here provide a summary of recent paleobiological studies that use FIB-EM methodology for the examination of fossils. Additionally, we demonstrate a novel method for analyzing the three-dimensional ultrastructure of microfossils (reported previously by Schiffbauer and Xiao [Palaios 24: 616–626, 2009]). This method, FIB-EM nanotomography, consists of sequential ion milling, or cross-sectioning, and concurrent SEM imaging, a technique that provides three-dimensional data of precise sites at high spatial resolution, yielding new insight into fossil ultrastructure. We here illustrate the use of FIB-EM nanotomography by studies of herkomorphic and acanthomorphic acritarchs (organic-walled microfossils) extracted from the ≥999 Ma Mesoproterozoic Ruyang Group of North China. The three-dimensional characteristics of important but controversial acritarch features such as extravesicular processes and vesicularly enclosed central bodies are described. Taken together, these case studies demonstrate that FIB-EM instruments are powerful and useful tools for investigating the three-dimensionality of microfossil ultra- and nanostructures.