This study describes the association between antibiotic resistance of bacteria causing neonatal bloodstream infection (BSI) and neonatal age to inform empirical antibiotic treatment guidelines. Antibiotic resistance data were analysed for 14 078 laboratory reports of bacteraemia in neonates aged 0–28 days, received by the Health Protection Agency's (now Public Health England) voluntary surveillance scheme for England and Wales between January 2005 and December 2010. Linear and restricted cubic splines were used in logistic regression models to estimate the nonlinear relationship between age and resistance; the significance of confounding variables was assessed using likelihood ratio tests. An increase in resistance in bacteria causing BSI in neonates aged <4 days was observed, which was greatest between days 2–3 and identified an age (4–8 days, depending on the antibiotic) at which antibiotic resistance plateaus to almost unchanging levels. Our results indicate important age-associated changes in antibiotic resistance and support current empirical treatment guidelines.