Very little is known about healthcare-associated infections (HAIs) in neonatal intensive care units (NICUs) in resource-limited settings including, India. The aim of this prospective study was to determine the prevalence, onset, risk factors and causative agents of laboratory confirmed blood stream (LBCI) as a HAI in a level-2 NICU at RD Gardi Medical College, Ujjain, India. The diagnosis of HAI was established using the Centre for Disease Control, USA criteria. A predesigned questionnaire containing risk factors associated with BSHAI was filled. A total of 150 neonates (43% preterm) were included in the study. The incidence of LBCI was 31%; 56% of which was late onset sepsis. The independent risk factors for LBCI were: preterm (Odds Ratio OR 3.22), duration of NICU stay more than 14 days (OR 2.38), chorioamnionitis in the mother (OR 18.48), neonate born through meconium stained amniotic fluid (OR 4.53), mal-presentation (OR 10.58), endotracheal intubation (OR 11.60), umbilical catheterization (OR 15.11), HAI due to ventilator-associated pneumonia (VAP) (OR 11.88). Initiation of minimal enteral nutrition was protective (OR 0.15). The predominant causative organisms were Gram-negative pathogens (58%). Among Klebsiella spp. and E. coli isolates, 73 and 80%, respectively were identified as extended-spectrum beta-lactamase producers. The results can be used to identify high-risk neonates for LBCI.