Nosocomial infection (NI) is a serious public health problem that affects hundreds of millions of people worldwide each year. The incidence of NI varied from 5.7 to 19.1% in developing countries and 3.6 to 12% in developed nations. In Rwanda, two studies conducted at two University Teaching Hospitals CHUK and CHUB revealed that the prevalence of nosocomial infections were 15.1% in 2013-2014 and 12.1% in 2015 at CHUK and CHUB respectively. Although the data are insufficient, they showed the high burden of the NI problem and the need to determine the incidence of NIs and the factors associated with them in Rwandan Hospitals. The main objectives of this study were to determine the incidence of NIs among adults patients in the medical wards of the University Teaching Hospital of Butare and identify the factors associated with acquisition of nosocomial infection. The study was carried out under a prospective follow-up study design in period of one month, all adults patients admitted in CHUB wards willing to participate were enrolled after signing an informed consent. During the study period, an infection arising at least 48 hours after admission was considered as NI. The sample size was raised to 206 participants obtained through a census sampling technique. The check list was used as data collection instrument to collect all related nosocomial infection information. The ethical requirements were considered. The incidence statistical formula, univariate, bivariate and multivariate analysis were processed. chi-square Test and logistic regression analysis were employed using SPSS version 21 considering the significance level of below 0.05 at 95%CI. The findings revealed that the overall incidence rate of nosocomial infections was 17.6 cases (95% CI: 16.7-18.4) per 1,000 person-days of hospitalization with the prevalence of 15.0%. Study subjects were followed for a total of 1827 person-days observation with a median time of 17.9 days (IQR = 12.7–23.6 days). The multivariate data analysis was conducted solely on the significant variables identified in the bivariate analysis to control for confounders. Table 4.8 illustrates that daily ward cleaning at or below one time was twelve times more likely to be associated with high nosocomial infection acquisition among hospitalized patients in CHUB, with an Adjusted odds ratio (AoR) of 11.613, Confidence Interval (CI): (3.679-43.467), and p<0.015. Bed pan insufficiency per patient and unavailability in the ward were also significantly associated with high nosocomial infection acquisition among hospitalized patients in CHUB, with AoR of 13.402 (CI: 1.879-27.438, p=0.01) and 4.664 (CI: 2.521-18.412, p<0.01), respectively. Healthcare Providers disinfect or not medical devices was associated with high nosocomial infection acquisition among hospitalized patients in CHUB, with an AoR of 12.095 (CI: 2.529-55.667, p=0.018). Cardiovascular diseases and diabetes were among the chronic diseases associated with high nosocomial infection acquisition among hospitalized patients in CHUB, with an AoR of 5.165 and 8.517 (CI: 1.103-14.020, 2.309-31.418); (p=0.03, p=0.02) respectively. This study concluded that inadequate ward cleaning, shortages of essential supplies, and the presence of chronic diseases was linked to the risk of acquiring a NI. Addressing these issues through enhanced cleaning protocols, disinfect medical devices, adequate supply management, and targeted care strategies for patients with underlying conditions is crucial for improving patient safety and healthcare outcomes.