Among the novel approaches applied to antimicrobial drug development, natural product-inspired synthesis plays a major role, by providing biologically validated starting points. Tetramic acids, a class of natural products containing a 2,4-pyrrolidinedione ring system, have attracted considerable attention for their antibacterial, antiviral, antifungal and anticancer activities. On the contrary, compounds with a 2,3-pyrrolidinedione skeleton have been considerably less investigated. In this work, we established chemical routes to the substituted 2,3-pyrrolidinedione core, which enabled the introduction of a wide range of diversity. In the perspective of a potential application for oral healthcare, a number of analogues with various substituents on the 2,3-pyrrolidinedione core were investigated for their antimicrobial and antifungal activities. The most promising compound showed a significant antimicrobial activity on Streptococcus mutans and Candida albicans, comparable to that of chlorhexidine, the gold standard in oral healthcare.