Four compounds with lactone moiety were synthesized from chalcone1in three- or four-step synthesis. γ-Bromo-δ-lactone5was the only product of bromolactonization of acid4whereas bromolactonization of ester3, apart from lactone5also afforded its isomer6and two diastereoisomeric δ-hydroxy-γ-lactones7and8. Lactone8was also obtained in 88% yield as a product of simultaneous dehalogenation and translactonization of γ-bromo-δ-lactone5byPenicillum frequentansAM 359. Chalcone-derived lactones5–8were subjected to the tests on antimicrobial activity and the results compared with activity of starting chalcone1. Obtained lactones5–8in most cases limited the growth of tested bacterial and fungal strains. The highest activity was found for δ-hydroxy-γ-lactone8which completely inhibited the growth ofStaphylococcus aureus, Fusarium graminearum,Aspergillus niger, andAlternariasp. The introduction of lactone moiety into chalcone scaffold significantly improved antimicrobial activity of the compound: γ-bromo-δ-lactone6and δ-hydroxy-γ-lactone8were significantly stronger growth inhibitors ofS. aureusandF. graminearum. In the case of the latter, a clear positive effect of the lactone function on the antifungal activity was also observed for γ-bromo-δ-lactone5.