Background: Testing of performance-associated parameters is a routine requirement in high-performance sports. Limited information is available regarding the anthropometrical, physiological and biomechanical characteristics of deaf soccer players. Objective: The primary purpose of this cross-sectional, descriptive study was to investigate possible performance-associated differences between the deaf Czech Republic national team soccer players and hearing counterparts from the Czech Republic First League. Methods: Seventy-four male soccer players (31 deaf and 43 hearing, ≥ 17 years of age) underwent laboratory testing that included measurement of autonomic cardiac activity, anthropometrical parameters, vertical jump performance, and maximal aerobic capacity via an incremental running test. Results: Compared with the hearing players, deaf players were shorter (p = 0.012), had more body fat (p < 0.001), a higher resting heart rate (p = 0.001), and ratio of Ln rMSSD to R–R interval length (p = 0.006), while maximal power output (p < 0.001), maximal oxygen uptake (p = 0.003), were significantly lower. After correcting for body fat percentage, the differences were not significant except for maximal power output value. Conclusions: Being at a disadvantage, the deaf soccer players differ from their hearing counterparts in various physiological or morphological parameters assumed to be associated with sports performance in soccer. However, the differences were minimized when taking 10% of body fat as a cut point into account. Thus, reduction in body fat percentage to the 10% level may be considered as one possible strategy for improving both the physical fitness and adaptability level of the studied deaf soccer players.