Intracellular bacterial pathogens have evolved sophisticated strategies to survive and proliferate within cells of their hosts. Studying their intracellular life cycle is key to understanding virulence and requires methodologies that can identify the compartments in which they localize and characterize the replicative niche they generate. Here, we describe immunofluorescence-based microscopy techniques applied to the intracellular pathogens Brucella abortus and Francisella tularensis during their respective intracellular cycles inside murine bone marrow-derived macrophages. Standard immunofluorescence techniques are used to define the intracellular localization of the pathogens based on their co-localization with specifically labeled macrophage organelles. In addition, we describe an assay to assess the integrity of Francisella-containing phagosomes and bacterial release into the macrophage cytoplasm, which is a hallmark of Francisella intracellular pathogenesis.