Introduction: The precise etiopathogenesis of the otitis media with effusion (OME) still remains unclear. Factors involved in the chronic inflammatory process in the mucosal lining of the middle ear cleft (which leads to accumulation of the middle ear fluid and consequently a hearing loss) are the dysfunction of the Eustachian tube, inapparent bacterial/viral infections and local allergic reactions. Patients and methods: Immunological and virological analysis of the middle ear effusion (MEE) samples collected intraoperatively from OME children were carried out. Children were examined with allergic tests. Concentrations of inflammatory proteins in the MEE were measured by nephelometry (albumin, immunoglobulin, complement-3 and -4, CRP) and ELISA (IL-4, IL-10, IFN-α, TNF-α). Reverse transcription – polymerase chain reaction – in situ hybridisation was used to detect rhino-enteroviruses in the effusion. HBoV was identified with quantitative PCR. Allergic tests consisted of the skin Prick-test and the measurement of the total- and allergen specific IgE concentration in the serum. Results: Based on the ratio of the concentrations of albumin and immunoglobulin-G in the effusion (A/G) samples were separated into two distinct groups characterised by different cytokine profile: proinflammatory cytokines (TNF-α, IFN-γ) dominate samples with low A/G, whereas inflammatory cytokines (IL-4, IL-10) are associated with high A/G value. One third of the samples (26 of the total 75) were virus positive. The prevalence of HBoV infection was as low as 2.7%. Mucoid samples were more frequently associated with virus infection. Allergy was established in one third of the children with Prick test. Allergen specific IgE positivity was more frequently diagnosed in the OME group than in the control group. Discussion: Accumulation of the MEE in the sample group characterised by high A/G value could be explained by transudation mechanism, where the increased capillary permeability is caused by vasoactive mediators from granulocytes stimulated by IL-4 and IL-10. Accumulation of MEE in the low A/G sample group could be a result of an active mucosal secretion which is party activated by proinflammatory cytokines produced consequently of the stimuli of viral nucleoproteins. The presence of the human respiratory viruses in the MEE and the frequent coincidence of allergy and OME give an evidence of the importance of these diseases in the etiopathogenesis of OME.