Objectives: Driveline infections remain an important complication of ventricular assist device therapy, with biofilm formation being a major contributor. This study aimed to elucidate factors that govern biofilm formation and migration on clinically relevant ventricular assist device drivelines. Methods: Experimental analyses were performed on HeartWare HVAD (HeartWare International Inc, Framingham, Mass) drivelines to assess surface chemistry and biofilm formation. To mimic the driveline exit site, a drip-flow biofilm reactor assay was used. To mimic a subcutaneous tissue environment, a tunnel-based interstitial biofilm assay was developed. Clinical HVAD drivelines explanted at the time of cardiac transplantation were also examined by scanning electron microscopy. Results: Common causative pathogens of driveline infections were able to adhere to the smooth and velour sections of the HVAD driveline and formed robust biofilms in the drip-flow biofilm reactor; however, Pseudomonas aeruginosa and Candida albicans had greater biomass. Biofilm migration within the interstitial driveline tunnel was evident for Staphylococcus epidermidis, Staphylococcus aureus, and C albicans, but not P aeruginosa. Biofilm formation by staphylococci was 500 to 10,000 times higher in the tunnel-based model compared with our exit site model. The 3-dimensional structure of the driveline velour and the use of silicone adhesive in driveline manufacturing were found to promote biofilm growth, and explanted patient drivelines demonstrated inadequate tissue in-growth along the entire velour with micro-gaps between velour fibers. Conclusions: This work highlights the predilection of pathogens to different parts of the driveline, the importance of the subcutaneous tunnel to biofilm formation and migration, and the presence of micro-gaps in clinical drivelines that could facilitate invasive driveline infections.