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The widespread use of intrapartum antibiotic prophylaxis (IAP) to prevent group B <i>streptococcus</i> (GBS) early-onset sepsis (EOS) is changing the epidemiology of EOS. Italian prospective area-based surveillance data (from 1 January 2016 to 31 December 2020) were used, from which we identified 64 cases of culture-proven EOS (<i>E. coli</i>, <i>n</i> = 39; GBS, <i>n</i> = 25) among 159,898 live births (annual incidence rates of 0.24 and 0.16 per 1000, respectively). Approximately 10% of <i>E. coli</i> isolates were resistant to both gentamicin and ampicillin. Five neonates died; among them, four were born very pre-term (<i>E. coli</i>, <i>n</i> = 3; GBS, <i>n</i> = 1) and one was born full-term (<i>E. coli</i>, n = 1). After adjustment for gestational age, IAP-exposed neonates had ≥95% lower risk of death, as compared to IAP-unexposed neonates, both in the whole cohort (OR 0.04, 95% CI 0.00–0.70; <i>p</i> = 0.03) and in the <i>E. coli</i> EOS cohort (OR 0.05, 95% CI 0.00–0.88; <i>p</i> = 0.04). In multi-variable logistic regression analysis, IAP was inversely associated with severe disease (OR = 0.12, 95% CI 0.02–0.76; <i>p</i> = 0.03). <i>E. coli</i> is now the leading pathogen in neonatal EOS, and its incidence is close to that of GBS in full-term neonates. IAP reduces the risk of severe disease and death. Importantly, approximately 10% of <i>E. coli</i> isolates causing EOS were found to be resistant to typical first-line antibiotics.