The PLL(PTE-like lactonase)-group of enzymes within the amidohydrolase superfamily hydrolyze N-acyl-homoserine lactones (AHLs) that are involved in bacterial quorum-sensing pathways. These enzymes possess the (beta/alpha)(8)-barrel fold and serve as attractive templates for in vitro evolution and engineering of quorum-quenching biological molecules that can serve as antivirulence therapeutic agents. Using a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 (GI: 41409766) as the initial template for in vitro evolution experiments, we enhanced the catalytic efficiency and increased the substrate range of the wild-type enzyme through a single point mutation on the loop at the C-terminal end of the eighth beta-strand. This N266Y mutant had an increased value of k(cat)/K(M) of 30- and 32-fold toward 3-oxo-N-octanoyl-l-homoserine lactone and N-hexanoyl-l-homoserine lactone, respectively; the evolved mutant also exhibited lactonase activity toward 3-oxo-N-hexanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone, AHLs that were previously not hydrolyzed by the wild-type enzyme. This article reinforces the evolutionary potential of the (beta/alpha)(8)-barrel fold and highlights the possibility of using quorum-quenching lactonases in the amidohydrolase superfamily as templates for engineering biomolecules of therapeutic use.