Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current study, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. We show that a number of antimicrobial compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-nonenyl)- quinoline (HMNQ) and long-chain rhamnolipid are present in or tightly associate with B. thailandensis OMVs. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against methicillin- resistant Staphylococcus aureus (MRSA). These findings indicate that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against biofilm- forming and multidrug-resistant organisms.