Masticatory muscle activity during teeth clenching is associated with changes in many physiological parameters throughout the body. Clenching can improve muscle activity, force production, rate of force development, and joint fixation. Hence, teeth clenching and masticatory muscle activity can be important in competitive sports activities. Sport climbing is becoming increasingly popular and will be included for the first time in the Summer Olympic Games, Tokyo, 2020. However, masticatory muscle activity in sport climbers has not yet been studied. The aim of the presented study is to compare the bioelectrical activity of the masticatory muscles in sport climbers and non-climbers in order to determine the relationship between these muscles and climbing activity. 44 subjects without masticatory system disorders (16 women and 28 men, average age 26.3) were divided into two groups of 22 sport climbers (8 women, 14 men, climbing experience >4 years), while 22 people (8 women, 14 men, with no regular sports activity) were assigned to the control group. Electromyographic examination of temporalis anterior (TA) and masseter muscle (MM) was evaluated in three conditions: during resting mandibular position, during maximum intercuspation clenching, and during maximum voluntary clenching with cotton rolls between teeth. For statistical analysis, the W Shapiro-Wilk test and the Mann-Whitney U test were used. Sport climbers showed significantly higher bioelectrical activities of MM during maximum intercuspation clenching (238.45 μV vs. 83.87 μV, p = 0.002), and during maximum voluntary clenching with cotton rolls between teeth (300.01 μV vs. 101.38 μV, p = 0.001) compared to controls. The differences between groups in relation to the resting bioelectrical activity of the MM muscles, and TA muscles in all conditions were not statistically significant (p > 0.05). Higher bioelectrical activity of masseter muscles during clenching in climbers can be associated with this sports activity. However, the mechanism remains unknown and requires future research.