BackgroundPatients already colonized with multidrug-resistant (MDR) Gram-negative bacteria (GNB) on admission to critical care units may be an important source of transmission of these bacteria in hospitals. We sought to determine the prevalence of MDR GNB colonization in patients, staff and the ward environment and to assess the risk factors for colonization of patients in wards. MethodsThe study was conducted from April 2021 to July 2021 in a teaching hospital in Ghana. MDR GNB were isolated from rectal, and hand swabs were taken from patients on admission and after 48 h. Swabs from HCW’s hands and the ward environment were also taken. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis. ResultsMDR GNB rectal colonization rate among patients was 50.62% on admission and 44.44% after 48 h. MDR GNB were isolated from 6 (5.26%) and 24 (11.54%) of HCW’s hand swabs and environmental swabs, respectively. Previous hospitalization (p-value = 0.021, OR, 95% CI= 7.170 (1.345–38.214) was significantly associated with colonization by MDR GNB after 48 h of admission. Age (21–30 years) (p-value = 0.022, OR, 95% CI = 0.103 (0.015–0.716) was significantly identified as a protective factor associated with a reduced risk of rectal MDR GNB colonization. ConclusionThe high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW’s hands, and the contamination of hospital environments highlights the need for patient screening and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals.