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Purpose Driveline infections remain a major complication of ventricular assist device (VAD) implantation. This study aimed to characterize in vivo microbial biofilms causing driveline infections and the degree of tissue integration into the velour of explanted drivelines. Methods Drivelines were obtained from ten VAD patients undergoing heart transplantation at the Alfred Hospital, Melbourne and St Vincent Hospital, Sydney between June 2017 to October 2018. Four uninfected drivelines and six infected drivelines were aseptically sectioned into 10 pieces of 1.5 cm in length, from the smooth tube section at the exit site to the velour section adjacent to the VAD. Organisms on each section were isolated and identified. Microbial biofilms on the infected driveline sections were assessed using scanning electron microscopy (SEM) and viable counts. Computed tomography (micro-CT) and SEM were used to assess tissue integration into each section. All sections were analysed histologically to confirm human matrix protein deposition. Results No organisms were isolated from non-infected drivelines using microbiological culture. Staphylococcus aureus, Pseudomonas aeruginosa , and Staphylococcus epidermidis were found on the velour sections of infected drivelines and correlated with the microbiological culture results from patient swabs of the infected exit site. Although histological analysis found considerable collagen fiber and fibroblast deposition on both the smooth tube and the velour, SEM and micro-CT suggested insufficient tissue integration throughout the driveline velour. Microgaps were observed between the velour fibres, with evidence of microbial biofilms within these gaps. Such biofilms were morphologically distinct from in vitro biofilms grown on biomaterials and might be responsible for the antimicrobial treatment failure. Conclusion This study demonstrated inadequate tissue integration of clinical drivelines in the subcutaneous tissue tunnel, with associated microbial biofilms formed within the microgaps between velour fibres. These data provide important insights into a potentially novel therapeutic strategy against driveline infections that is focused on enhancing tissue integration into the velour, thereby preventing microbial adherence, biofilm formation and migration. [ABSTRACT FROM AUTHOR]