Healthcare-associated infections (HAI’s) caused by nosocomial pathogens are accountable for high morbidity and mortality. The aim of this study was to conduct the surveillance of nosocomial pathogens in clinical laboratory departments in hospitals of Ha’il region. In this study, 255 samples were collected, of which 84% were positive for nosocomial pathogens. The total microbial isolates among the positive samples were 844 (average 3.31 bacteria per sample). The results of this study showed a high percentage of multi-drug resistant (MDR) bacteria. From the total S. aureus collected isolates, the methicillin resistant S. aureus (MRSA) was found to be 66% from the hospital environments; 55% among healthcare staff, and 42% among healthcare students. Furthermore, a high percentage of extended-spectrum beta-lactamase (ESBL) positive Escherichia coli, Klebsiella species, and Enterobacter cloacae was detected. From the environmental samples, the ESBL rate among Escherichia coli, Klebsiella species, and Enterobacter cloacae was found to be 60%, 75%, and 70% respectively. On the other hand, from healthcare-staff the ESBL rate among Escherichia coli, Klebsiella species, and Enterobacter cloacae was found to be 50%, 60%, and 50% respectively. The results of this study conclude a high rate of pathogenic organisms from various study sites (labs, HCWs, HCSs, environment, and control subjects). This gives a strong indication for the possibility of transferring other organisms, which may cause very serious problems to hospital staff as well as the community. To prevent microbial contamination, standard guidelines must be followed, and infection control strategies should be developed and implemented.