- Background: The widespread use of 7-valent pneumococcal conjugate vaccine (PCV7) has changed acute otitis media (AOM) bacteriology. Only scattered data with regard to this effect of PCV13 have been published so far. Methods: We retrospectively identified children <6 years of age who presented to our hospital with AOM, and had middle ear fluid (MEF) cultures obtained during tympanocentesis or from spontaneous otorrhea during 2008–2013, when PCV7 (2009) and PCV13 (2010) were gradually introduced in the Israeli National Immunization Program. Data were extracted for demographics, clinical and microbiologic parameters, according to vaccination status. Results: Of the 295 eligible AOM episodes reported in 279 children, 224 (76%) had MEF cultures from tympanocentesis and 71 (24%) from spontaneous otorrhea. Boys and children <2 years of age contributed 178 (60%) and 219 (74%) AOM episodes, respectively. Acute mastoiditis complicated 58 (20%) of these episodes. None of the children were PCV immunized in 2008, but >90% had received ≥1 PCV dose(s) by 2011 or later. Of the 106 (36%) MEF cultures which tested positive for otopathogens, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and multiple bacteria grew in 60 (57%), 39 (37%), 2 (2%) and 5 (5%) episodes, respectively. S. pneumonia-positive MEF culture rate in unimmunized children (31, 69%) was significantly higher than in PCV7-immunized children (22, 59%) or PCV13-immunized children (12, 50%), P = 0.04 and P = 0.02, respectively. Conclusion: PCV7 and PCV13 implementations in the Israeli National Immunization Program were associated with a rapid reduction of “severe” pneumococcal AOM episodes.