Enteral nutrition via a percutaneous endoscopic gastrostomy (PEG) tube is often part of management in patients with dysphagia due to neurological or oropharyngeal disease. Gastrostomy placement can affect normal innate defense mechanisms in the upper gut, resulting in bacterial overgrowth. In this study microbiological investigations were done with gastric and duodenal aspirates from 20 patients undergoing PEG tube placement and PEG tubes from 10 patients undergoing tube replacement. Aspirate and PEG tube microbiotas were assessed by using viable counts and selective solid media followed by aerobic and anaerobic incubation to assess cell viabilities. The antibiotic susceptibility profiles of the isolates were determined by the disk diffusion method, and gas chromatography was used to study the bacterial metabolic products in the aspirates. The aspirates and PEG tubes contained mainly streptococci, staphylococci, lactobacilli, yeasts, and enterobacteria. Enterococci were detected only in PEG tube biofilms and not in aspirates. Gastric pH affected the composition of the aspirate microbiotas but not the total microbial counts. Staphylococci, Escherichia coli, and Candida spp. were isolated only from antibiotic-treated patients, despite the sensitivities of the bacteria to the agents used. Antibiotic treatment had no effect on the incidence of infection or the length of hospital stay in these patients.