The surface brightness fluctuation (SBF) method is a robust and efficient way of measuring distances to galaxies containing evolved stellar populations. Although many recent applications of the method have used space-based imaging, SBF remains a powerful technique for ground-based telescopes. Deep, wide-field imaging surveys with subarcsecond seeing enable SBF measurements for numerous nearby galaxies. Using a preliminary calibration, Cantiello et al. presented SBF distances for 89 bright, mainly early-type galaxies observed in the Next Generation Virgo Cluster Survey. Here we present a refined calibration and SBF distances for 278 galaxies extending several magnitudes fainter than in previous work. The derived distances have uncertainties of 5%–12% depending on the properties of the individual galaxies, and our sample is more than 3 times larger than any previous SBF study of this region. Virgo has a famously complex structure with numerous subclusters, clouds, and groups; we associate individual galaxies with the various substructures and map their three-dimensional spatial distribution. Curiously, subcluster A, centered around M87, appears to have two peaks in distance: the main peak at ∼16.5 Mpc, and a smaller one at ∼19.4 Mpc. Subclusters B and C have distances of ∼15.8 Mpc. The W and W ′ groups form a filament-like structure, extending more than 15 Mpc behind the cluster with a commensurate velocity increase of ∼1000 km s−1 along its length. These measurements are a valuable resource for future studies of the relationship between galaxy properties and local environment within a dynamic and evolving region.