Cyclophilins are a family of proteins which exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity. They catalyse the cis to trans isoform change of xaa-proline bonds which is considered a rate limiting step of protein folding. Cyclophilins have been associated with virulence in intracellular bacteria such as Brucella abortus and Legionella pneumophila, in particular playing a role in the ability of the bacterium to adhere to surfaces. Due to their highly conserved nature, cyclophilins present as anti-virulence targets for many pathogenic bacteria, such as Burkholderia pseudomallei, which encodes two cyclophilin genes, ppiA and ppiB. B. pseudomallei is the causative agent of melioidosis, which is endemic in South-East Asia and Northern Australia. Even with appropriate antibiotic treatment the mortality rate of melioidosis in Australia is still ~15%. Due to rising levels of antibiotic resistance and a lack of effective treatment for melioidosis, novel medical countermeasures are urgently needed. This study investigates the potential for cyclophilins as novel anti-virulence targets in B. pseudomallei. To understand the role of cyclophilins in B. pseudomallei, in-frame ppiA and ppiB deletion mutants were constructed and characterised. Cyclophilins appear to play a role in the adhesive capability of both B. pseudomalleiΔppiA and B. pseudomalleiΔppiB mutants and display decreased biofilm formation. Furthermore both deletion mutants demonstrate increased susceptibility to stress particularly to oxidative stress. Ultimately resulting in decreased ability to invade murine macrophages, indicating they play an important role in virulence. Therefore, cyclophilins represent novel class of anti-virulence targets in B. pseudomallei.