Patients with dysphagia require long-term nutritional support. This can be delivered by the enteral route via a percutaneous endoscopic gastrostomy (PEG) tube. Enteral nutrition (EN) bypasses the body's innate defences that prevent the microbial colonization of the proximal gut, which predisposes to microbial overgrowth. A continuous culture model simulating the upper gastrointestinal tract microbiota of EN patients was used to investigate the effects of a synbiotic (Lactobacillus acidophilus DUN-311, Bifidobacterium bifidum BB-02, Bifidobacterium lactis BL-01, Synergy 1) on microbial community structure and metabolism. A PEG tube was inserted into the fermenters to study biofilm formation. The synbiotic delivered in sterile semi-skimmed milk (SSSM) was introduced either 48 similar to h prior to or after PEG tube insertion. The synbiotic reduced biofilm formation on PEG tube surfaces, with suppression of Escherichia coli and Klebsiella pneumoniae when it was added subsequent to PEG insertion. When synbiotic feeding was commenced prior to PEG insertion, colonization by Staphylococcus aureus, Candida albicans and Candida famata was also inhibited. Lactate production increased in response the synbiotic or control (SSSM). These results indicate that the use of a synbiotic has the potential to reduce pathogen colonization on PEG tube surfaces in vivo, thereby reducing the incidence of biofilm-related infectious complications.