Microorganisms play important roles in intravascular catheter (IVC)-related infections, which are the most serious complications in children with IVCs, leading to increased hospitalisation, intensive care admissions, extensive antibiotic treatment and mortality. A greater understanding of bacterial communities is needed in order to improve the management of infections. We describe here the systematic culture-independent evaluation of IVC bacteriology in IVC biofilms. Twenty-four IVC samples (six peripherally inserted central catheters, eight central venous catheters and ten arterial catheters) were collected from 24 paediatric patients aged 0 to 14 years old. Barcoded amplicon libraries produced from genes coding 16S rRNA and roll-plate culture methods were used to determine the microbial composition of these samples. From a total of 1,043,406 high-quality sequence reads, eight microbial phyla and 136 diverse microbial genera were detected, separated into 12,224 operational taxonomic units (OTUs). Three phyla (Actinobacteria, Firmicutes and Proteobacteria) predominate the microorganism on the IVC surfaces, with Firmicutes representing nearly half of the OTUs found. Among the Firmicutes, Staphylococcus (15.0 % of 16S rRNA reads), Streptococcus (9.6 %) and Bacillus (6.1 %) were the most common. Community composition did not appear to be affected by patients’ age, gender, antibiotic treatment or IVC type. Differences in IVC microbiota were more likely associated with events arising from catheter dwell time, rather than the type of IVC used.