Purpose: The aim of this study was to examine whether collagenase producing bacteria could be detected, in fluid collected from chest tubes, before clinical presentation of anastomotic leakage after esophageal resection. Methods: We conducted a prospective single-center study of patients who underwent resection of the gastroesophageal junction. All patients had a chest tube placed in the pleural cavity perioperatively. Drain fluid was collected and cultured from the first post-operative day and at time of routine removal of the drain (days 3–5). Results: From January 2018 to July 2019, a total of 84 patients were included in the study. Twenty (36%) patients experienced severe complications with a Clavien-Dindo score of 3b-5. Eleven (13%) patients were diagnosed with anastomotic leakage which occurred after 8 days (mean, range 2–13). Twenty patients (24%) had drain samples with significant growth of microbes. Among the 11 patients with anastomotic leakage, we found 2 with microbe growth at POD 2 and POD 4, the remaining 9 samples were negative (p = 0.638). Thirty-day mortality rate was zero. Conclusion: Cultured fluid from the pleural cavity of asymptomatic patients following esophageal resection did not indicate a significant association with anastomotic leakage.