Sepsis is one of the major causes of neonatal death worldwide as well as in Bangladesh. The objective of the present study was to identify the risk factors and causative organisms of neonatal sepsis after delivery in a tertiary care hospital, Bangladesh. This was a case-control study conducted in the neonatal ward of Rajshahi Medical College Hospital (RMCH), a 1000-bed tertiary hospital situated in Rajshahi, Bangladesh. Neonates diagnosed as neonatal sepsis by clinical and laboratory parameters were included as cases in this study. Admitted neonates unsuspected or undiagnosed for sepsis were considered as controls. Maternal and neonatal information and their laboratory reports were collected and analyzed. Both bivariate and multiple logistic regression models were used to identify the risk factors of neonatal sepsis. A total of 91 cases and 193 controls were included in the study. Maternal history of urinary tract infection (UTI) during the third trimester of pregnancy (aOR 2.75, 95% CI: 1.04-7.23, p <0.05), premature birth (aOR 2.77, 95% CI: 1.08-7.13, p <0.05) and APGAR score <7 at five minutes (aOR 2.58, 95% CI: 1.04-6.39, p <0.05) were associated with onset of neonatal sepsis in multiple logistic regression model. All these factors were also associated with developing early-onset neonatal sepsis, while maternal UTI and male sex of neonates were associated with developing late-onset neonatal sepsis. Escherichia coli (40.7%), Staphylococcus aureus (27.5%), and Klebsiella pneumoniae (18.7%) were the commonly isolated organisms causing neonatal sepsis. All these organisms were highly resistant to common antibiotics like amoxicillin, cephalosporins, aminoglycosides and quinolones. Carbapenemase group of drugs along with amikacin, nitrofurantoin and linezolid were the most sensitive drugs. Strengthening the existing facility for antenatal screening for early diagnosis and treatment of maternal infection during pregnancy as well as identifying high-risk pregnancy for adequate perinatal management is necessary to prevent neonatal sepsis-related morbidity and mortality. Rational use of antibiotics according to local epidemiology and culture and sensitivity reports may minimize the increasing hazards of antibiotic resistance.