The gastrointestinal tract is a crucial battleground in microbial pathogenesis, where pathogens must adhere to, invade, and translocate through intestinal epithelial cells to establish infection and cause disease. Investigating these processes is critical for understanding the mechanisms of infection and host defense. In vitro assays using intestinal cell models enable precise analysis of microbial adhesion, invasion, and translocation at the cellular level. In contrast, in vivo, rodent models provide valuable insights into the complex interactions between pathogens, host tissues, and immune responses. Advanced imaging techniques, such as confocal microscopy, offer high-resolution visualization of host–pathogen interactions, allowing researchers to observe pathogen-induced alterations to the intestinal epithelial barrier and study microbial dissemination. By combining functional assays with cutting-edge imaging technologies, researchers can uncover critical mechanisms that drive microbial pathogenesis, revealing how pathogens breach intestinal defenses and spread systemically. This integrated approach deepens our understanding of infection dynamics and facilitates the development of new therapeutic strategies to combat gastrointestinal infections. Here, we describe a comprehensive approach to studying microbial pathogenesis by integrating in vitro and in vivo assays of intestinal cell adhesion, invasion, and translocation alongside advanced imaging techniques, such as confocal microscopy using immunostaining and fluorescence in situ hybridization (FISH) in tissue sections, to elucidate host–pathogen interactions and infection dynamics at the cellular level.