Background: Septicaemia is the third most common cause of neonatal death after prematurity and birth asphyxia. The prevalence of neonatal sepsis and the spectrum of causative microorganisms fluctuates over time, thus facility-specific surveillance of neonatal bloodstream infections is important. Increasing levels of antimicrobial resistance documented worldwide, necessitate regular monitoring of institutional resistance patterns to ensure appropriate and effective empirical antimicrobial therapy. Method:The laboratory blood culture reports and patient records from a neonatal unit at a Botswana referral hospital were retrospectively reviewed to determine the one-year prevalence of neonatal sepsis and the antimicrobial susceptibility patterns of common pathogens. Results: Of 909 neonates investigated for suspected sepsis using 1 119 blood cultures, 89 (9.8%) had laboratory-confirmed episodes of bloodstream infection (septicaemia). The most prevalent pathogens included Klebsiella pneumoniae (29.4%), group B streptococcus (16.3%) and Escherichia coli (11.9%). Blood culture contamination rates were high at 18.6% (208/1 119). Gramnegative pathogens showed low susceptibility to gentamicin (40%) and cefotaxime (47%), but high susceptibility to amikacin (86%). Streptococci and enterococci were moderately sensitive to ampicillin (79%), and fully susceptible to vancomycin. Methicillin-resistant Staphylococcus aureus was not isolated. Exposure to maternal syphilis and previous antibiotic exposure were significantly associated with neonatal septicaemia. Conclusion: Neonatal sepsis is common, with a predominance of Gram-negative pathogens. The high rate of blood culture contamination should be addressed. Emerging antibiotic resistance may require clinicians to review currently used antibiotics for the empirical treatment of late-onset neonatal septicaemia.