Ear infection or otitis is a generic term used to refer to ear infection or inflammation. The purpose of the present study was to compare culture and Multiplex PCR methods to identify Alloiococcus otitidis, Moraxella catarrhalis, and Haemophilus influenza as three fastidious pathogens causing otitis. 100 patients, who had the physician-confirmed infection of the external ear and otitis media, were randomly sampled. The samples were analyzed with culture and Multiplex PCR. None of the three bacteria were found in samples of otitis externa by culture or PCR. In the case of otitis media three isolates of Alloiococcus otitidis, one isolation of Moraxella catarrhalis, and three strains of Haemophilus influenza were found using culture method. In the case of otitis media three isolates of Alloiococcus otitidis, one isolation of Moraxella catarrhalis, and three strains of Haemophilus influenza were found using culture method, but the rate of these bacteria were 25, 11 and 28 isolates using Multiplex PCR. The Multiplex PCR method was found to be furthermost sensitive, truthful and reproducible screening method for detection of fastidious bacteria.