- Background Because the microbiology and susceptibility patterns of middle ear fluid pathogens in children with otitis media change over time, an active surveillance is recommended to establish appropriate therapeutic guidelines. Objective To analyze the microbiology and susceptibility pattern of middle ear pathogens obtained from Costa Rican children with acute otitis media (AOM), recurrent otitis media (ROM) and therapeutic failure otitis media (OMTF) between 2002 and 2007. Patients and methods 1108 children aged 2–92 months who participated in various otitis media clinical trials between the years 2002 and 2007. Results Among the study population, 880 were children with AOM (61% <24 months of age), 138 were children with ROM (54% <24 months of age) and 90 were children with OMTF (67% <24 months of age). Bilateral otitis media was more frequent in children with OMTF (44%) than in children with AOM (37%) (P = 0.19) and ROM (27%) (P = 0.009). Presence of siblings <8 years of age was more frequently observed in children with OMTF (73%) than in children with ROM (65%) (P = 0.0001) and AOM (47%) (P = 0.000002). Overall Streptococcus pneumoniae (44%) was the most common pathogen isolated followed by Haemophilus influenzae (37%), Moraxella catarrhalis (11%) and Streptococcus pyogenes (4%). S. pneumoniae was the most common pathogen in AOM (44%) and ROM (47%), however, H. influenzae was the most common pathogen in OMTF (40%). Among all H. influenzae, an increase in the number of β-lactamase producing strains was observed from 5.2% in 2001 to 14% (P = 0.04) in 2007 and this was associated with an increase in the use of amoxicillin. An increase in the number of M. catarrhalis was also observed, from 3% (9/350) in 2001 to 11% (71/628) (P = 0.000003) in 2007. During the study period the incidence of penicillin non-susceptible S. pneumoniae was 42/211 (20%) in children with AOM; 5/35 (17%) in children with ROM and 5/17 (42%) in children with OMTF. M. catarrhalis cases increased from 8% in 2004 to 17% in 2007 (P = 0.0005) and S. pyogenes decreased from 7% in 2002–2004 to 1% in 2005–2007 (P = 0.001). Conclusions In Costa Rica, S. pneumoniae remains the most common pathogen in children with AOM and ROM whereas non-typable H. influenzae remains the most common pathogen in children with OMTF. A significant increase in the number of β-lactamase positive H. influenzae and M. catarrhalis has been observed in recent years.