Background: Every year, more than 17.7 million endoscopic procedures take place. Although endoscopic procedures are generally low-risk, multidrug-resistant bacteria have been identified as a contributing factor to endoscope-associated infection(EAI). From here, the value of microbiological surveillance is gradually becoming a challenge to determine the best practice for endoscope surveillance. Methods: We aim to compare various guidelines for microbiological surveillance of endoscopes. Different types of endoscopes are sampled after undergoing high-level disinfection (HLD) by the conventional flushing sampling method (CFSM), flush-brush-flush sampling method (FBFSM), and rapid protein test. Different brush heads were used while sampling by the FBFSM. Results: There was a high significant difference between the two used methods, being the highest among FBFSM and the lowest among CFSM, with detection ranges (2–72 cfu) and (0–30 cfu), respectively, and the FBFSM were able to isolate more high concern bacteria than the CFSM. Additionally, a pull-through brush when added to the sampling process of FBFSM, improved the culture outcomes significantly. Conclusion: FBFSM was confirmed to be more sensitive than CFSM in bacterial detection and the usage of the pull-through brush in the case of FBFSM increased its sensitivity in the detection of microorganisms. Microbiological surveillance can't be replaced by rapid protein test.