The transport of micrometer- and submicrometer-sized particles of biotic and abiotic origin through both natural and engineered porous media is relevant to a wide variety of disciplines. While the influence on colloid transport of chemical factors such as colloid and media surface chemistry and the ionic strength of the carrying fluid are fairly well understood, the role of physical factors still needs clarification from a mechanistic standpoint. Along with recent recognition that pore-scale processes may depend on the coupling of chemistry and local hydrodynamics, which in turn depends on pore topography, there is a growing need for experimental systems that enable visualization of individual colloids in pores and pore networks that contain relevant three-dimensional features. We introduce a single camera–epifluorescence microscope system to track fluorescent colloids in three dimensions using the apparent diameter of out-of-focus colloids to estimate their z-axis location. The current system, using a Plan Achromat 5× objective, enables z estimation of 4.7-μm-diameter fluorescent microspheres across a range of 1200 μm with accuracy of approximately 34 μm. In addition to discussion of system limitations and potential improvements, we present results from two mini-studies illustrating the use of three-dimensional particle tracking to investigate (i) the behavior of colloids approaching orthogonally oriented grain-to-grain contacts, and (ii) the potential retention mechanisms in porous media under chemical conditions unfavorable to deposition. The ability to track individual colloids in three dimensions brings experimental capabilities closer in line with computer-modeling capabilities, setting the stage for synergistic pairing of experimental and theoretical investigations.