- Background: Pathogen eradication in patients with acute otitis media (AOM) is associated with a reduced risk of clinical failures, but most children in whom middle ear fluid (MEF) culture remains positive show clinical improvement or clinical cure. We investigated the relationship between MEF culture-positivity during treatment in patients with clinical improvement/cure, and the occurrence of subsequent AOM. Methods: A total of 673 patients with culture-positive MEF were enrolled in double-tympanocentesis studies and followed for 3 weeks after completion of treatment. Results: On day 4–6, 189/673 (28%) patients had culture-positive MEFs. Patients with clinical improvement/cure on day 11–14 (end of treatment) despite having culture-positive MEF on day 4–6 more often had recurrent AOM episodes (53/151, 35%) than those with culture-negative MEF (114/476, 24%; P = 0.007). 41/53 (77%) culture-positive patients with clinical improvement/cure on day 11–14 underwent tympanocentesis when AOM recurred and 29/41 (71%) were culture-positive. Pulsed field gel electrophoresis identity between pathogens at recurrence and those persisting on day 4–6 was found in 19/29 (66%) compared with 31/86 (36%) of the evaluable patients with recurrence and culture-negative MEF on day 4–6 (P = 0.005). Conclusions: (1) Failure to eradicate MEF pathogens during antibiotic treatment is associated with clinical recurrences, even in patients showing clinical improvement/cure at end of treatment; (2) these recurrences are mostly caused by pathogens initially present in MEF and persisting during treatment.