Objective To study whether humidifier reservoir bacteria would colonize other parts of the breathing circuit with the help of airflow during mechanical ventilation. Methods Totally, 30 ICU patients on invasive mechanical ventilation therapy were selected. Samples were collected from the patients′ humidifier reservoir water, and reservoir outlet and sent for bacterial culture. Then 30 experimental models were established to simulate mechanical ventilation in vitro, with Pseudomonas aeruginosa being inoculated into humidifier reservoir water of 15 models, and Acinetobacter baumannii into the rest of the models. Then broth was added into each reservoir to boost bacterial reproduction. At 24 h, 72 h, and 168 h after inoculating the bacteria, samples were taken from the humidifier reservoir water and from different sites of the circuit and sent for bacterial culture. Results Among the 30 clinical samples, the culture positivity rate of bacteria was 46.7% in the humidifier reservoir water, and 16.7% at the reservoir outlet. Among the 30 in vitro experimental models, the bacteria in the humidifier reservoirs grew in large amount, and the culture positivity rate of bacteria at 24 h, 72 h, and 168 h after inoculation was not statistically significant (P>0.05). The culture positivity rate of bacteria at the reservoir outlet was 36.7% at 24 h after inoculation, 63.3% at 72 h, and 76.7% at 168 h, with significant difference (P<0.05). However, bacterial culture at the Y-shaped circuit and at 10 cm from the reservoir outlet 168 h after inoculation was negative. At 72 h, 168 h after inoculation, bacterial concentration at the reservoir outlet in the Pseudomonas aeruginosa group was significantly higher than in the Acinetobacter baumannii group (P<0.05 for both). Conclusion During mechanical ventilation, there will be bacterial colonization in the humidifier reservoir water. If bacteria grow in large amount in the humidifier reservoir, they can colonize the outlet of the reservoir with the help of airflow, and the amount of bacteria colonizing the outlet of the reservoir varies by types of bacteria.