The increasing prevalence of nosocomial pathogens in hospital environment has become a serious concern with significant morbidity, mortality and high cost burden on society and health care systems worldwide. This increase has been mainly associated with increased health care-associated infections (HAIs), a surge of antibiotic use and new developments in life saving medical practices. The problem is further complicated by the emergence of difficult to treat multidrug-resistant (MDR) micro-organisms. In order to control and reduce the prevalence of nosocomial pathogens within healthcare settings, it becomes imperative to acquire the knowledge about the source and transmission of these difficult to treat pathogens. The aim of this study was to screen the patients, staff and the environment of Maternity Hospital in northern part of Saudi Arabia. In this study, 878 swabs were collected from a range of body sites from patients, staff and from the hospital environment like walls, beds, taps, computer keyboards and ventilator surfaces. The isolated bacterial strains were identified by conventional methods as well as by automated methods, including MALDI-TOF-MS and Microscan. Furthermore, the antimicrobial susceptibility was performed by Microscan. In this study, 157 different bacterial strains were isolated including, S. aureus (32.48%), E. coli (19.74%), K. pneumoniae (32.48%), Enterobacter spp. (5.73%), Citrobacter (0.63%) and P. aeruginosa (8.9%). Among these bacterial isolates, 41.4% were found to be in staff, 56.6% in patients and 1.9% in hospital environment respectively. The antibiotic susceptibility results revealed that, 57% and 43% of MRSA were found among staff members and patients respectively. All (100%) Extended spectrum â-lactamase (ESBL) positive E. coli and 91% ESBL positive K. pneumoniae were found among patients respectively. The findings of current study showed a high percentage of nosocomial pathogens prevalent in Maternity Hospital in Ha’il region of Saudi Arabia. Additionally, a high percentage of multi drug resistant strains from both patients and staff members further stresses on the importance of surveillance and effective infection control strategies to be implemented among the hospitals and healthcare facilities in Saudi Arabia.