BackgroundGroup B Streptococcus (GBS) that asymptomatically colonizing the recto-vaginal area of women is the most important cause of neonatal colonization. There is paucity of evidence about newborn colonization with GBS in Ethiopia. Thus, this study was aimed to determine the prevalence of newborn colonization with GBS, antibiotic susceptibility patterns of the isolates and associated risk factors at the University of Gondar Referral Hospital in Northwest EthiopiaMethodsA prospective cross sectional study was conducted from December 2016 to November 2017. A total of 1,155 swabs from nasal, ear and umbilical areas of the newborns were collected from the 385 newborns. Identifications of the isolates and antibiotic susceptibility testing were done by using conventional methods.ResultsSixty two (16.1%, 95% CI: 12.2% - 20%) of the newborns were colonized by GBS. Seven percent of the total specimens were positive for GBS. The antibiotics susceptibility rates of GBS (average of the three body sites tested) were 95.1%, 89.6%, 88.9%, 85.7%, 85.3%, 81.3%, 76.9%, 76.1%, 73.8%, and 34.4% to ampicillin, penicillin, ciprofloxacin, chloramphenicol, vancomycin, azitromycin, erythromycin, clindamycin, ceftriaxone, and tetracycline, respectively. A multilogistic regression analyses were shown that the newborns that were from mothers whose education status was below tertiary level, and newborns from mothers who were: being employed, being nullipara and multigravida were at risk for colonization with GBS.ConclusionPrevalence of neonatal colonization with GBS was higher than it was reported in three decades ago in Ethiopia. Ciprofloxacin, chloramphenicol, vancomycin and azithromycin were identified as the drug of choice next to ampicillin and penicillin.Electronic supplementary materialThe online version of this article (10.1186/s12887-018-1350-1) contains supplementary material, which is available to authorized users.