The Gram-negative bacteria Klebsiella pneumoniae can cause severe infections in elderly, diabetics, and immunocompromised individuals, such as urinary tract infections (UTI), pneumoniae, and sepsis. Many UTIs are associated with indwelling urinary catheters since K. pneumoniae is able to colonize medical equipment; this is related to the fact that the pathogen is also able to create biofilms on catheters. The creation of biofilms on abiotic surfaces is also seen in other bacteria, such as Escherichia coli, from which it is known that amyloid fibers constitute most of the biofilm and it contributes to the formation of it. Amyloids in different bacteria have been identified, yet none have been characterized in K. pneumoniae strains. Thus, the aim of this study is to characterize a new K. pneumoniae amyloid in biofilm formation. To identify the amyloid, bacterial cultures with Congo red (CR) were made to create a biofilm. The biofilm was then assessed with brightfield- and fluorescence microscopy, and the fibers in this biofilm were characterized by electron microscopy (EM). Subsequent protein identification was performed by SDS-PAGE, in-gel digestion, MALDI-TOF MS and MS/MS. Of the seven K. pneumoniae isolates that were cultivated, isolate CA402 exhibited the most prominent results in biofilm formation, binding of CR and brightfield- and fluorescence microscopy, all displaying distinctive traits of amyloid fibers; CA402 was therefore used for the rest of the experiments. EM also displayed fibers with characteristic properties of amyloid, indicating these are amyloid fibers found in the biofilm. SDS-PAGE of the amyloid fibers that were treated with formic acid showed a distinctive band at 20kDa, indicating it could contain the amyloid protein. Four interesting peptide masses appeared after measuring with MALDI-TOF, but the peptide mass at 1510.738 m/z was the most distinctive. Subsequent MS and MS/MS revealed that this peptide matched with the MrkA subunit of type 3 fimbriae in a CA402 protein database. This indicates that the amyloid fiber found in K. pneumoniae CA402 is MrkA. The findings thereby provide a better understanding of the pathogenesis, as type 3 fimbriae are known to play a role in biofilm formation and MrkA is part of the type 3 fimbriae