The aim of this study was to compare incidence, bacteriology and associated mortality of neonatal sepsis in a neonatal unit, after (2001-2004) and before (1995-1996) implementation of universal screening for prevention of Group B Streptococcus diseases. Early onset sepsis incidence decreased from 2.5 to 1 per 1000 live births (p = 0.03), with a decrease in the proportion of Group B Streptococcus cases from a 54% to 11% (p < 0.01). In late onset sepsis, coagulase-negative Staphylococci continued to be the predominant pathogen (49% of cases), and Candida albicans emerged as the second etiologic agent in the postscreening period. Sepsis associated mortality was low in both periods (2.2%).