Antimicrobial resistance (AMR) constitutes a major public health crisis with antibiotic resistant bacterial infections taking 700,000 of lives each year. Emerging AMR is due to the misuse of antibiotics. Unlike antibiotics, anti-virulence drugs aim to disarm bacteria by inhibiting their virulence factors without affecting growth. Disulfide bond formation (Dsb) is critical for protein stability and the catalyst DsbA is considered a promising anti-virulence target as its inhibition will simultaneously inactivate several virulence factors that bacterial pathogens employ during infection. Some pathogenic bacteria, like uropathogenic Escherichia coli (UPEC) encode multiple Dsb proteins, but their contribution to biogenesis of different virulence factors is unclear. Our study has investigated the role of DsbA homologues in UPEC growth and virulence in physiologically relevant in vitro conditions. We showed that DsbA enzymes are not important for aerobic growth of UPEC strain CFT073 in LB medium. However, in M9 minimal medium, the lack of dsbA but not dsbL showed significantly reduced UPEC growth. Furthermore, we have shown that DsbA enzymes are essential for functional type 1 fimbriae and flagella expression in UPEC grown in rich and minimal media. Our findings support the notion that inhibition of DsbA will disarm UPEC during host infection and also likely slow down bacterial growth in the urinary tract. Future work aims to assess the full antimicrobial potential of DsbA inhibitors as novel therapeutics for antibiotic-resistant urinary tract infections.