The ultrasonic cleaning method was found to be the most effective method for the removal of biologic waste when compared with the manual method with a nylon brush. However, there was no significant difference between these two methods when using enzymatic detergent. Both the manual method and the ultrasonic method showed significantly higher effectiveness when enzymatic detergent was used for the cleaning of Flexoreamer K-type files, although it is evident that neither method achieved complete removal of biologic waste from all Flexoreamer K-type files, which could lead to improper sterilization of endodontic files and, consequently, be a factor to be considered in clinical cross-infection.