Neonatal group B streptococcal (GBS) infections are one of the important health problems because of their high mortality and morbidity rates in certain countries. There are some preventive approaches, including perinatal antibiotic therapy against these infections. Recently, vaccination with conjugated GBS polysaccharides has also been practised. In this study, the in vitro inhibitory effects of 51 lactobacilli (of them 50 were purified from vaginal swabs, 1 from a commercial vaginal tablet) on five GBS (4 clinical isolates and 1 standard strain) were investigated by sandwich plate technique and deferred antagonism well technique. Ten clinical isolates (20%) and the drug-purified Lactobacilli expressed pronounced inhibitory effects on growth of GBS. All of the inhibitory isolates and 10 randomly selected non-inhibitory isolates were identified by API 50CHL kit (BioMeriéx, France). Seven (70%) of the inhibitory clinical isolates were Lactobacillus rhamnosus. The inhibitory isolates had higher acid production than the non-inhibitory ones (p < 0.05), and pH-adjustment destroyed their inhibitory effects entirely. If these results could be applied in vivo, it could be postulated that administration of certain lactobacilli as probiotics via an appropriate regimen may be a safe, physiological and cheaper alternative for prevention of neonatal GBS infections.