Abstract To evaluate in vitro the influences of various moisturizing pretreatment approaches on the cleaning effect and mechanical properties of small reusable dental instruments. The mostly used instruments in the dental clinic, such as surgical high-speed long fissure burs, slow-speed round burs and Ni-Ti root canal instruments, were selected to analyze the effects of the pretreatment methods. In the control group, water was utilized as the moisturizer. In the experimental groups, DR. WEIGERT multi-enzyme cleaner (enzyme group) and PRE-Klenz instrument transport gel (professional moisturizer group) were used for moisturizing pretreatment, and each instrument was reused four times. The cleanliness of the instruments was observed under an optical microscope magnified 25 times and scored. Rapid Adenosine Triphosphate (ATP) testing was used to measure surface cleanliness of the instruments. Simultaneously, the surface contaminant residue and mechanical wear of instruments after last recycling were observed under a scanning electron microscope (SEM) and analyzed by the energy dispersive spectroscopy (EDS). The results showed that the professional moisturizer group demonstrated superior effects in terms of cleanliness and ATP residue detection compared with the enzyme cleaner and water groups (P < 0.05). Moreover, during the recycling of the instruments, the professional moisturizer group maintained a stable cleaning effect. The results of SEM indicated that the instruments in the professional moisturizer group had less surface contaminant residue, and the results of EDS also revealed less mechanical wear. This study provides an experimental foundation and clinical guidance for moisturizing pretreatment methods of dental instruments.