Objective To compare the differences of two sampling tools used for active screening on nasal bacterial carriage of health care workers (HCWs), evaluate colonization of multidrug-resistant organisms (MDROs) in the nasal cavity of HCWs in department of neurosurgery, and provide basis for the prevention and control of MDROs. Methods In July 2019, 110 HCWs in department of neurosurgery of a hospital were selected as the research objects, their nasal bacterial carriage was actively screened. 0.85% normal saline sampling tube (sponge swab) was used for the left nostril of HCWs, disposable nasal swab (cotton swab) was used for right nostril, sampling result of two tools was compared. The isolated MDROs were performed drug resistance analysis and decolonization, bundle intervention measures were taken in the ward, effect before and after the intervention was compared. Results There are 110 HCWs, including 31 doctors, 66 nurses, 7 attendants, 6 further-traning students and normative training of resident physicians participated in study. A total of 46 strains of bacteria were isolated, 10 of which were MDROs, isolation rates of methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Acinetobacter baumannii (CRAB) were 8.18% and 0.91% respectively, carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa, and vancomycin-resistant Enterococcus were not found. The specimens collected by two sampling methods were cultured for 24 hours, 48 hours and 72 hours, there were no significant differences in colony detection and semi-quantitative results between two groups (both P>0.05); there were significant differences in the sampling time, success rate of single collection, loss rate of consumables and staff's satisfaction between two sampling methods (all P<0.05). Cotton swab method was better than sponge swab method, and the cost was lower. Nine strains of MRSA had high resistance to erythromycin and clindamycin, drug susceptibility spectrum of 2 strains isolated from nurses were identical. After taking preventive and control measures, 8 HCWs with positive MRSA all turned negative, infection rate and isolation rate of MDROs, including MRSA and CRAB in wards decreased compared with those before intervention (P<0.05). Conclusion Colonization rate of MRSA in the nasal cavity of HCWs in department of neurosurgery is high, detection results of bacteria with normal saline sampling tube and disposable nasal swab are almost the same, from the comprehensive consideration of various aspects, especially cost-effectiveness, cotton swab is better than sponge swab. It is suggested to monitor bacterial colonization in nasal cavity of HCWs in key departments, timely de-colonize, and strengthen the comprehensive prevention and control measures with contact isolation as the core measure, so as to effectively reduce the incidence of MDROs colonization rate in HCWs.