The aim of the study was to determine the prevalence of vaginal group B streptococcus (GBS) colonization in pregnant women from Osijek area, the possible effect of GBS colonization on pregnancy outcome and neonatal complications and the role of intrapartum prophylaxis in this context. This retrospective case-control study took place at the Department of Gynecology and Obstetrics, Osijek University Hospital Center from December 2003 to June 2006. A total of 118 pregnant women was enrolled in study and divided into two groups: 59 women in 35th-37th week of gestation, free from risk factors for infection (control group); and 59 women in 25th-41st week of gestation with risk factors for infection. Low vaginal swab for GBS isolation and identification on selective and enriched medium was obtained from each woman. GBS colonization was recorded in 29 (24.6%) women: 12 (20.3%) control and 17 (28.8%) women at risk of infection, yielding a statistically non-significant difference (Chi2 = 1.480489; p < 0.48). Early neonatal infection was observed in six (20.7%) neonates born to 29 mothers with GBS colonization, pointing to a correlation between vaginal GBS colonization and early neonatal infection (r(s) = 0.99). Early perinatal infection was found in 22 (18.6%) neonates, including 17 (28.8%) pregnancies with risk factors, pointing to a significant correlation between vaginal GBS colonization, risk factors and early perinatal infection (Chi2 = 88.68; p < 0.001); however, gestational age and pregnancy outcome were not influenced by GBS colonization. In eight (36.4%) newborns, early neonatal infection developed in spite of intrapartum administration of antibiotics; three of these children were born to GBS positive mothers, and perinatal GBS infection was demonstrated in one (0.84%) child. Study results revealed a relatively high rate of GBS colonization in the population of pregnant women in Croatia, occasionally leading to early neonatal infection. Large studies are needed to develop national strategy for the prevention of GBS infection in Croatia.