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Macrophages are critical to the formation of infection- and non-infection-associated immune structures such as cancer spheroids, pathogen-, and non-pathogen-associated granulomas, contributing to the spatiotemporal and chemical immune response and eventual outcome of disease. While well established in cancer immunology, the prevalence of using three-dimensional (3D) cultures to characterize later-stage structural immune response in pathogen-associated granulomas continues to increase, generating valuable insights for empirical and computational analysis. To enable integration of data from 3D in vitro studies with the vast bibliome of standard two-dimensional (2D) tissue culture data, methods that determine concordance between 2D and 3D immune response need to be established. Focusing on macrophage migration and oxidative species production, we develop experimental and computational methods to enable concurrent spatiotemporal and biochemical characterization of 2D versus 3D macrophage–mycobacterium interaction. We integrate standard biological sampling methods, time-lapse confocal imaging, and 4D quantitative image analysis to develop a 3D ex vivo model of Mycobacterium smegmatis infection using bone-marrow-derived macrophages (BMDMs) embedded in reconstituted basement membrane (RBM). Comparing features of 2D to 3D macrophage response that contribute to control and resolution of bacteria infection, we determined that macrophages in 3D environments increased production of reactive species, motility, and differed in cellular volume. Results demonstrate a viable and extensible approach for comparison of 2D and 3D datasets and concurrent biochemical plus spatiotemporal characterization of initial macrophage structural response during infection.