Port wine vinegar, a product of the esteemed Port wine, is renowned for its intricate blend of flavors and aromas, a result of complex microbial interactions. This study delves into the fascinating world of yeast and acetic acid bacteria (AAB) interactions during fermentation, which significantly influence the vinegar’s chemical composition and sensory properties. We specifically investigate the role of yeasts in fermenting sugars into ethanol, a process that AAB then converts into acetic acid. The impact of these interactions on the production of secondary metabolites, such as gluconic acid, ketones, aldehydes, and esters, which contribute to the vinegar’s unique sensory profile, is thoroughly examined. Advanced analytical techniques, including GC-MS and e-nose technology, alongside sensory evaluation, are employed to assess these effects. The research underscores the significance of ethanol tolerance in AAB and other production challenges in determining vinegar quality and underscores the importance of optimizing fermentation conditions and sustainable practices. The findings of this study underscore the importance of strain interactions and production techniques, which can significantly enhance the quality and market appeal of Port wine vinegar, providing valuable insights for the industry. This review also identifies exciting and critical areas for future research, inspiring further exploration and proposing strategies for advancing production and application in culinary, health, and industrial contexts.