About four million deaths occur every year worldwide due to neonatal sepsis, of which 99% take place in low-income countries. Nearly 71% of neonatal deaths are preventable and among these, 10% are due to neonatal sepsis. Nevertheless, limited information exists on neonatal sepsis and its associated factors in Rwanda. The objectives of the study were to find prevalence and factors associated with neonatal sepsis among neonates admitted in Kibungo Referral Hospital, Ngoma District, Rwanda. A retrospective cross-sectional study design was used to recruit all neonates admitted at Kibungo Hospital in 2017. Neonates related records were reviewed to collect data. Logistic regression and Odds ratios were calculated to identify the factors associated with neonatal sepsis at 95% CI, p < 0.05. A total of 422 neonates were enrolled in the study, 12.8% (n = 54) had neonatal sepsis, and 62% of blood culture revealed Klebsiella pneumoniae. Among neonates with neonatal sepsis, 38 (70%) recovered while 16 (30%) died. Strong association with neonatal sepsis was found with neonatal age less or equal to three days (AOR: 2.769, 95%; CI, 1.312–5.843; P = 0.008); and gestational weeks less than 37 weeks (aOR: 4.149; CI: 1.1878–9.167; p = < 0.001). Mothers and hospital environments should be monitored to prevent infection through the regular blood culturing and ward decontamination.