Objective To compare the disinfection effect of alcohol pads and cotton swabs on needleless infusion connectors of peripheral venous catheters, and provide reference for clinical selection of appropriate disinfection tools. Methods Patients with peripheral venous catheters for≥48 hours in departments of respiratory medicine, nephrology/endocrinology, and gastroenterology in July-September 2024 were selected. In stage 1, three types of swabs were adopted to collect specimens of cross-sectional and threaded surfaces of undisinfected needleless infusion connectors from 12 patients at 24, 48, and 72 hours of retention. Cotton swabs were selected as the final sampling tool. In stage 2, a self-controlled and randomized crossover design was adopted. Needleless infusion connectors of 65 patients were disinfected by alcohol pads and alcohol cotton swabs at different time points. In the first group, after intravenous catheters maintained retention for 48 hours and 72 hours, the needleless infusion connectors were disinfected by alcohol pads and alcohol cotton swabs, respectively. In the second group, after the intravenous catheters maintained retention for 48 hours and 72 hours, the needleless infusion connectors were disinfected by alcohol cotton swabs and alcohol pads, respectively. The research subjects were divided into two groups according to the random number table method, and specimens were taken after disinfection. All data on alcohol pad disinfection were summarized as data for alcohol pad group, and data on alcohol cotton swab disinfection were summarized as data for alcohol cotton swab group. The microbial conditions on the surface of needleless infusion collectors, duration of disinfection operation, and nurses’ satisfaction were compared between the alcohol pad group and the alcohol cotton swab group. Results The microbial positive rates in alcohol pad group were both 0 at 48 and 72 hours, those in alcohol cotton swab group were 3.33% and 3.23%, respectively, without statistically significant difference between two groups (both P>0.05). The duration of disinfection operation in alcohol pad group at 48 and 72 hours were (21.71±3.84) and (21.28±3.01) seconds, respectively, while that in alcohol cotton swab group were (31.39±4.03) and (33.45±3.85) seconds, respectively, with statistically significant differences between two groups (both P<0.05). Nurses’ satisfaction with alcohol pad disinfection was significantly higher than that with cotton swabs (100% vs 60.00%, P<0.05). Conclusion Although there is no statistically significant difference in the disinfection effect of alcohol pads and cotton swabs on needleless infusion collectors, alcohol pads save disinfection time and improve nurses’ satisfaction, which can be promoted and applied in clinical practice.