Background <p>The most important step in endoscope retreatment is careful manual cleaning before disinfection. Selecting the proper brushes is critical to disinfection. The study investigated the effectiveness of a novel double-headed disposable brush for cleaning flexible gastrointestinal endoscopes.</p> Methods <p>This prospective, quasi-experimental study included endoscopes used in a first-class hospital between January 2020 and August 2021. The primary outcome was the change in the amount of bacteria determined using the relative light unit (RLU) value of adenosine triphosphate (ATP) fluorescence.</p> Results <p>A total of 267 endoscopes (131 double-headed brushes and 136 conventional ones) were included. The changes in RLU values of the ATP detection before and after brushing in the double-headed group were significantly higher than in the conventional group (ΔRLU, 116 (55, 251) vs. 76 (26, 181), <i>P</i> = 0.001). Similar results were observed in the subgroup analysis of colonoscopes, especially those with a 3.8-mm channel (ΔRLU, 115 (34, 265) vs. 52 (19, 199), <i>P</i> = 0.007), but not for gastroscopes (<i>P</i> = 0.363). Positive bacterial culture rates were higher in the double-headed/nylon brush end and double-headed/non-woven fabric end than in the single-headed brush (40.5% and 70.2% vs. 33.1%, <i>P</i> &lt; 0.001).</p> Discussion <p>The results indicated the advantage of the novel double-headed disposable brush for flexible endoscope cleaning, and the nylon brush end may explain the advantage.</p> Conclusion <p>The cleaning effect of the novel double-headed disposable brush for the flexible endoscope was better than the conventional one.</p> Trial registration <p>Not applicable.</p>