Background: Sepsis is a major cause of mortality in neonates in sub-Saharan Africa. Bloodstream infections (BSIs), the most common hospital-associated infections in neonates, occur more frequently in resource-limited countries than in industrialized countries and contribute to many in-hospital neonatal deaths. We assessed the efficacy of a novel bundle of low-cost infection prevention and control (IPC) measures targeted to known and suspected risk factors for neonatal hospital-associated BSI. We studied the impact of this intervention bundle on BSI and mortality in a neonatal intensive care unit (NICU) at the University Teaching Hospital (UTH) in Lusaka, Zambia. Methods & Materials: This prospective observational cohort study of neonates who survived > 3 days after NICU admission consisted of a six-month baseline period (“baseline”), two months during which interventions were introduced (“implementation”), and 10 months of intervention assessment (“intervention”). The intervention bundle consisted of IPC training, introduction of alcohol-based hand wash, weekly bathing of neonates > 1.5 kg with 2% chlorhexidine, targeted environmental cleaning, and SMS reminders of IPC messages. The outcomes were all-cause neonatal mortality (primary) and suspected sepsis and laboratory-confirmed BSIs (secondary). Results: From September 2015 through March 2017, we enrolled 2669 eligible neonates. Median maternal age, maternal HIV status, and newborn characteristics were similar in the baseline, implementation, and intervention periods (with the exception of birth weight). Half of neonates had one or more episodes of sepsis, including 549 (41%) who had a positive blood culture. Klebsiella pneumoniae, predominantly ESBL-producing, was most common (n = 289, 70%); possible contaminants were isolated in most other positive cultures (n = 140, 25.5%). All-cause neonatal mortality was lower during the intervention (18.0%) than the baseline period (23.6%), and similar reductions in mortality were seen in all birthweight categories. The incidence density rates for suspected sepsis and BSI with a pathogen were significantly lower in the intervention relative to baseline periods for all birth weight categories, except babies weighing < 1 kg. Conclusion: This bundle of infection prevention measures resulted in reductions in all-cause neonatal mortality, suspected sepsis, and BSI with pathogens. This combination of low-cost measures has potential to be applied in other contexts where hospital-associated sepsis is a major contributor to neonatal mortality.