Microsporidia are obligate intracellular pathogens that infect a wide range of invertebrate and vertebrate hosts, including humans. In humans, microsporidia most often infect the gastrointestinal tract and cause diarrheal diseases, but in immunocompromised patients, infections can be fatal. Previous knowledge of microsporidian intracellular development was based on 2D transmission electron microscopy images and light microscopy. This study used serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis, within host cells. The dataset includes SBF-SEM, live-cell imaging, and light microscopy data. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection.