Background: Nosocomial infection is an infection that hospitalized patients get while receiving health care. This study aimed to determine the prevalence and factors involved in the incidence of nosocomial infections, related microbial agents, and antibiotic resistance profiles. Methods: This cross-sectional study was conducted on 390 patients with nosocomial infection admitted to Shahid Beheshti Hospital in Sabzevar from 2011 to 2015. The acquired data were assessed by the standard checklist of the National Nosocomial Infections Surveillance (NNIS) system of the Ministry of Health. The collected data were analyzed using the Stata 12 software. Results: Of the 41979 admitted patients during the mentioned period, 390 patients with an average age of 48.08 years suffered from a hospital-acquired infection. The prevalence of nosocomial infections was 1% with the highest rate related to the respiratory tract in men and surgical sites in women. Intravenous catheters, surgical wounds, urinary catheters, suction, ventilator, tracheotomy, and tracheostomy were the most commonly encountered factors (invasive measures) of nosocomial infection, respectively. The most prevalent causes of nosocomial infections were Klebsiella, Staphylococcus aureus, and Acinetobacter. Conclusions: Based on our findings, several factors play a pivotal role in preventing hospital-associated infections, including proper follow-up and timely reporting of nosocomial infection cases. The other influential factors were accurate identification of microorganisms involved, allocation of sufficient funds to provide appropriate facilities for infection prevention, the appropriate and timely medical interventions, and the rational use of antibiotics.