The analysis of movement patterns through EMG activity provides the opportunity to identify the muscle groups most involved in a particular exercise, and to determine the scope of inter-limb deficiencies. The aim of the present study was to investigate the effects of a side-to-side muscle activity asymmetry between the left and the right lower limb during sprinting in soccer players. Sixteen professional soccer players took part in the study. Their age, body mass and body height equaled 23.7 ± 7.6 years, 81.2 ± 10.8 kg and 179.3 ± 12.2 cm, respectively. The sprint test consisted of two maximal sprints over 30 m with a 5-min rest interval between each sprint. EMG was recorded bilaterally from the quadriceps, hamstrings and gluteal muscles. Regression analysis revealed a significant effect of a side-to-side average muscle activity asymmetry between the left and right hamstring (LH/RH) muscles during the speed tests at 5 m (p = 0.044), and 30 m (p = 0.045), as well as the left and right glutes (LG/RG) at 5 m (p = 0.044) and 30 m (p = 0.043). Our results indicate that hamstring and glute muscles should be selectively and additionally activated during resistance training in soccer players to prevent injuries and improve sprint performance.