Abstract Background Catheters are the most common cause of nosocomial infections and are associated with increased risk of mortality, length of hospital stay and cost. Prevention of infections and fast and correct diagnosis is highly important. Methods In this study traditional semiquantitative culture-dependent methods for diagnosis of bacteria involved in central venous catheter-related infections as described by Maki were compared with the following culture-independent molecular biological methods: Clone libraries, denaturant gradient gel electrophoresis, phylogeny and fluorescence in situ hybridization. Results In accordance with previous studies, the cultivation of central venous catheters from 18 patients revealed that S. epidermidis and other coagulase-negative staphylococci were most abundant and that a few other microorganisms such as P. aeruginosa and K. pneumoniae occasionally were found on the catheters. The molecular analysis using clone libraries and sequencing, denaturant gradient gel electrophoresis and sequencing provided several important results. The species found by cultivation were confirmed by molecular methods. However, many other bacteria belonging to the phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were also found, stressing that only a minor portion of the species present were found by cultivation. Some of these bacteria are known to be pathogens, some have not before been described in relation to human health, and some were not closely related to known pathogens and may represent new pathogenic species. Furthermore, there was a clear difference between the bacterial species found in biofilm on the external (exluminal) and internal (luminal) side of the central venous catheter, which can not be detected by Maki's method. Polymicrobial biofilms were observed on most of the catheters and were much more common than the cultivation-dependent methods indicated. Conclusion The results show that diagnosis based on molecular methods improves the detection of microorganisms involved in central catheter-related infections. The importance of these microorganisms needs to be investigated further, also in relation to contamination risk from improper catheter handling, as only in vivo contaminants are of interest. This information can be used for development of fast and more reliable diagnostic tools, which can be used in combination with traditional methods.