Group A Streptococcus (GAS) is a significant human pathogen responsible for severe diseases in over 18 million individuals annually, with more than 500,000 deaths worldwide. Despite its considerable global impact, there is currently no available vaccine. Additionally, rising concerns about reduced penicillin sensitivity and increasing macrolide resistance emphasize the urgent need for alternative therapeutic strategies. PrsA, a lipid-anchored extracellular peptidyl-prolyl isomerase (PPIase), is essential for stabilizing and folding secreted proteins at the membrane-cell wall interface and plays a key role in the pathogenesis of Gram-positive bacteria. In GAS, two isoforms, PrsA1 and PrsA2, have been identified. Our earlier research demonstrated that these isoforms are highly conserved across GAS serotypes, are crucial for virulence factor secretion, and can elicit protective immune responses when combined with complete Freund's adjuvant. In this study, we found that PrsA1 and PrsA2 predominantly localize to detergent-resistant membrane microdomains, forming discrete foci within the membrane. Deletion of prsA in GAS resulted in significant alterations to membrane protein composition, the formation of aggregates, reduced bacterial survival, and increased susceptibility to membrane-targeting antimicrobial agents. These findings suggest that PrsA is a potential target for the development of novel therapeutic approaches.