Information on pneumococcal vaccination's impact on the prevention of acute otitis media (AOM) at very young ages is limited. To define the trends in tympanocentesis-proven AOM incidence, clinical characteristics, microbiology, and antibiotic resistance in infants <2 months of age in southern Israel, before and after the sequential introduction of 7- and 13-valent PCVs. A retrospective population-based cohort study including children <2 months of age diagnosed with AOM at the pediatric emergency room between January 2005-Decmber 2009 (pre-vaccination group, group 1) and January 2013-July 2021 (post-PCV13 introduction, group 2). 160 patients were enrolled, 89 (55.6%) in group 1 and 71 (44.4%) in group 2. The mean incidence of AOM decreased from 1.2 cases/1000 live births for group 1 to 0.45 cases/1000 live births for group 2, P<0.001.130 (81.25%) patients were hospitalized, with higher hospitalization rates in group 1 vs. group 2 (84/89, 94% vs. 46/71, 65%, P<0.001). Hospitalization length was longer in group 1 vs. group 2 (4.07±4.09 days vs. 2.70±1.82 days, P=0.021). Positive MEF cultures were reported in 94/160 (58.75%) patients, with a decrease in positivity rates between the 2 groups (71/89, 80% vs. 23/71, 32%, P<0.001). S. pneumoniae was the most common pathogen (55/94, 58.5%); it was the most frequent pathogen isolated in group 1 (46/71, 65%), and the second most common pathogen in group 2 (9/23, 39%), P=0.03. A significant increase was recorded in the percentages of patients with negative MEF cultures (from 21% to 68%, P<0.001). The introduction and implementation of PCV13 in southern Israel was associated with a decrease in AOM in children <2 months of age and of S. pneumoniae recovery in these patients and was accompanied by less admissions and shorter hospitalizations. An increase in the proportions of negative bacterial cultures from MEF was recorded during the study period.