Objective The purpose of this investigation was to assess the muscular strength of the knee extensors and flexors muscles, agility and functionality between dominant leg (DL) vs. non-dominant leg (NDL) of professional soccer players during pre-season. Methods Twenty-two healthy male professional soccer players (25.1 +/- 3.8 years; 182.1 +/- 5.9 cm; 79.3 +/- 5.4 kg; body fat 14.6 +/- 3.8%) from a club of the Brazilian second-division soccer league participated in this study. All soccer players underwent three tests to assess strength, power, and physical performance with an interval of 48 h inter-sessions. During the first assessment session, isokinetic tests were performed in which athletes performed maximal isokinetic concentric and eccentric contractions, as well as maximal isometric contractions. In the second and third assessment sessions, single leg step down and agility tests were performed, respectively. Results Maximum voluntary isometric contraction (MVIC) results showed a significant difference (p < 0.04) between DL compared to the NDL for absolute and relative values. But, the isokinetic variables of knee extensors at 60 degrees/s (total work and power) were significantly higher (p < 0.03) in NDL. Pearson's correlation was found to be significant between MVIC and single leg step down (SLSD) test for DL (r = 0.70, p = 0.0001) and NDL (r = 0.58; p = 0.002). Conclusion The results confirm that DL shows greater isometric strength than NDL. However, NDL revealed an increase in the total work and power output in professional soccer players. Furthermore, the SLSD test is recommended because it proved to be a good method for evaluating muscle strength by a positive correlation with MVIC.