Objective To provide theoretical basis for determining whether disinfection is necessary before reco-nnecting the infusion device after intermittent administration of medicine via needleless connectors (NC) during intravenous infusion. Methods The actual situation of clinical intravenous infusion was simulated in a laboratory environment. Taking intravenous injection as an example, microorganisms on the surface of NC and in the remaining liquid after infusion at different intermittent administration times (1-9 times) were detected by adenosine triphosphate (ATP) bioluminescence detection technique. The experiment was repeated in 15 groups for each administration frequency, and a total of 270 specimens were detected and statistically analyzed. ATP detection value ≤25 relative light unit (RLU) was considered to be qualified. Results With the increase of intermittent administration frequency, the overall microorganisms on NC surface gradually increased, but all were ≤25 RLU. Microorganisms on the surface of NC among different disconnection times were statistically different (P<0.001). Compared with the microorganisms when disconnecting 9 times, numbers of microorganisms on the surface of NC with disconnection of 1, 2, 3, and 4 times were all statistically different, respectively (all P<0.05), while those with other disconnection frequencies did not show statistical differences among each other (all P>0.05). Microorganisms in liquid after infusion among different intermittent administration frequencies were not statistically different (P=0.946). Conclusion In laboratory environment, with the increase of intermittent administration frequency, microorganisms on the surface of NC increase, but the safety of the infusion process is not compromised.