The purpose of this study was to describe the variation in listeriosis infection incidence during pregnancy over a 25-year period based on salient clinical and laboratory features compiled in a tertiary referral hospital and to depict the clinical characteristics of these cases. A cohort was created of all cases of listeriosis in pregnant women or their neonates (early-onset form) diagnosed between 1985 and 2010. Forty-three cases of perinatal listeriosis were diagnosed among the 82,320 hospitalised pregnant women (incidence 0.5‰). Whereas the incidence remained almost constant at 0.24‰ until 2000, an increasing incidence was observed from then on, reaching 0.86‰ during the last years of the study period. A four-fold increase in listeriosis rate during pregnancy has occurred in recent years, with poorer outcome for those cases occurring before 28 weeks and for those in which early antibiotic treatment was not provided. These results should raise the awareness of the agencies and professionals involved in prenatal care.