Xylella fastidiosa encodes multiple toxin-antitoxin systems, including dinJ/relE and mqsR/ygiT. Phylogenetic analyses indicate these two toxin-antitoxin systems have distinct evolutionary histories. Comparisons among X. fastidiosa subspecies/strains reveal toxin-antitoxin systems are often embedded in prophage sequences, suggesting acquisition via horizontal transfer. Tagged proteins of both toxin-antitoxin systems were over-expressed, purified, and evaluated for activity. Toxins MqsR and RelE are ribonucleases. MqsR cleaved at G(triangle)CU and G(triangle)CC; RelE lacked specificity in vitro. YgiT and DinJ antitoxins inhibit activity of the cognate toxin by direct binding. Single (toxin or antitoxin) and double (toxin and antitoxin) knock-out mutants were constructed in X. fastidiosa strain Temecula. Both antitoxin mutants displayed reduced planktonic cell density relative to wild type. The dinJ(-) mutant produced less biofilm, whereas the ygiT(-) mutant produced more biofilm, relative to wild type. Planktonic cell density of both toxin mutants was unaffected, with biofilm reduced slightly. Planktonic cell density and biofilm formation of double mutants were similar to wild type. However, viable cell counts for planktonic cells and biofilm of double mutants were more than one order of magnitude greater than wild type. Collectively, the results indicate that dinJ/relE and mqsR/ygiT are functional toxin-antitoxin systems and likely play different roles in regulation of X. fastidiosa population levels. Published by Elsevier Ltd.