Summary: Background: The early-onset sepsis calculator (EOSC) reduces unnecessary antibiotic treatment in newborns. However, its performance in identifying cases with early-onset disease (EOD) is unclear. We compared the sensitivity of the EOSC to the current Dutch and National Institute for Health and Care Excellence (NICE) guidelines when applied to a cohort of newborns with culture-positive early-onset sepsis and meningitis. Methods: Culture-positive Streptococcus agalactiae (GBS) and Escherichia coli (E. coli) sepsis and meningitis patients ≤3 days old with a gestational age ≥34 weeks, identified between 1/1/2018 and 31/1/2021 in a Dutch prospective nationwide cohort study were included. Cases were identified by treating physicians and microbiological surveillance. Primary outcome was the proportion of patients that would have been treated according to the EOSC, the Dutch, and the NICE EOD prevention guidelines. Differences between proportions were analysed using McNemar's test. Findings: We included 81 GBS and 7 E. coli EOD cases. At 4 h after birth, the EOSC would have recommended antibiotic treatment in 32 (36%) patients, compared to 44 (50%) by the Dutch (p<0·01) and 48 (55%) by the NICE guideline (p<0·01). The EOSC would have initially recommended routine care for 52% of patients compared to 31% and 30% for the Dutch and NICE guidelines (p<0·01). At 24 h after birth, the EOSC would have recommended antibiotic treatment in 54 (61%) infants compared to 64 (73%) by the Dutch (p = 0·02) and 63 (72%) by the NICE guidelines (p = 0·06). Interpretation: The sensitivity of the EOSC in identifying cases of EOD is lower compared to both Dutch and NICE guidelines, especially directly after birth. The EOSC relies more on clinical symptoms and results in less overtreatment of healthy newborns at the cost of later antibiotic treatment in initially well-appearing EOD patients. Funding: This work was supported by grants received from Netherlands Organization for Health Research and Development (ZonMw; ...