Harboring four transmembrane domains in their structural hallmark, Tetraspanins (Tspans) are a family of glycoproteins with pivotal functions in a variety of biological and cellular processes. Through interacting laterally with each other or specific membrane proteins, Tspans organize tetraspanin-enriched microdomains (TEMs), modulating cellular signaling, adhesion, fusion, and proliferation. An abundance of evidence has identified the multiple functions in the progression of cancer as well as the underlying molecular mechanisms. Recently, plenty of studies have focused on the utilities of Tspans by pathogens for infection, especially the infection of viruses. The expression of Tspans correlates with the phase of viral infection, the type of virus, and targeted therapies. In particular, perturbations of Tspans in host cells can affect viral attachment, intracellular trafficking, translation, virus assembly, and release. In this review, we summarize and provide a historical overview of the discovery and characterization of various kinds of virus infection and highlight their diversity and complexity, along with the virus life cycle. Furthermore, we examined the current understanding of how various Tspans are involved in the regulatory mechanisms underlying viral infection. This review aims to offer a comprehensive understanding of the targeting of Tspans for therapeutic intervention in infections caused by diverse pathogens.