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The advent of human intestinal organoid systems has revolutionized the way we understand the interactions between the human gut and microorganisms given the host tropism of human microorganisms. The gut microorganisms have regionality (ie, small versus large intestine) and the expression of various virulence factors in pathogens is influenced by the gut milieu. However, the culture conditions, optimized for human intestinal organoids, often do not fully support the proliferation and functionality of gut microorganisms. In addition, the regional identity of human intestinal organoids has not been considered to study specific microorganisms with regional preference. In this review we provide an overview of current efforts to understand the role of microorganisms in human intestinal organoids. Specifically, we will emphasize the importance of matching the regional preference of microorganisms in the gut and tailoring the appropriate luminal environmental conditions (ie, oxygen, pH, and biochemical levels) for modeling real interactions between the gut and the microorganisms with human intestinal organoids.