Digital holographic microscopy is an ideal tool for investigation of microbial motility. However, most designs do not exhibit sufficient spatial resolution for imaging bacteria. In this study we present an off-axis Mach-Zehnder design of a holographic microscope with spatial resolution of better than 800 nm and the ability to resolve bacterial samples at varying densities over a 380 μm × 380 μm × 600 μm three-dimensional field of view. Larger organisms, such as protozoa, can be resolved in detail, including cilia and flagella. The instrument design and performance are presented, including images and tracks of bacterial and protozoal mixed samples and pure cultures of six selected species. Organisms as small as 1 μm (bacterial spores) and as large as 60 μm (Paramecium bursaria) may be resolved and tracked without changes in the instrument configuration. Finally, we present a dilution series investigating the maximum cell density that can be imaged, a type of analysis that has not been presented in previous holographic microscopy studies.