Opportunistic pathogens are one of the most frequent causes of hospital infections in children intensive care units (ICU). The aim of our research was to estimate the colonization and infection with opportunistic pathogens in patients, personnel and the environment in Tartu and Tallinn ICU. A control card for hospital infection surveillance (CCHIS) was elaborated. During the nine-month period of the surveillance study, bacteriological samples were collected from patients. At the beginning and at the end of the surveillance study a prevalence study (samples isolated from the environment and personnel) was performed. The study group comprised 422 patients with 1873 samples, 88 personnel members with 471 samples and 195 samples from the ward environment. The isolated microbial strains were investigated pheno- (antibiotic susceptibility) and genotypically (PFGE). According to the data from CCHIS no differences were detected in the structure of the patients’ age and sex in Tartu and Tallinn ICUs. In both wards coagulase-negative staphylococci (CONS) and K. pneumoniae were isolated most frequently. Hospitalization period was longer in patients colonised with MRSE or K. pneumoniae than in non-colonised patients. In Tallinn MRSE and in Tartu, ESBL producing K. pneumoniae presented a problem. According to molecular methods the MRSE strains isolated from the blood of newborns were multiclonal and differed genotypically from the MRSE strains isolated from the personnel. Most of the ESBL producing K. pneumoniae, isolated from patients, belonged to two clones that were not detected in the environment. We conclude that different multiresistant microbial strains caused hospital infections in the studied ICUs. Unfortunately, we did not manage to detect the origin of the circulating MRSE and ESBL producing K. pneumoniae.