The aim of this study was to test the hypothesis that high levels of testosterone during prenatal life, testified by a low second-to-fourth digit ratio (2D:4D), as well as in adulthood affect the aggressive behavior of professional soccer players. Using 18 male professional players from a first level Italian Soccer Team we calculated: i) the 2D:4D ratio of the right hand, ii) the number of yellow and red cards per game, iii) the mean salivary testosterone concentration (Sal/T) and iv) the handling of aggressive impulses as assessed by the Picture Frustration test (PFT). Soccer players with a lower 2D:4D ratio had a higher number of fouls per game. A significant negative correlation was observed between Sal/T and 2D:4D ratio, as well as between 2D:4D ratio and the aggressiveness of players. By contrast, a significant positive correlation of Sal/T and fouls/game score and PFT was detected. No significant correlation was detected between 2D:4D or Sal/T and the playing position of players. Results of this study revealed that in professional soccer players, aggressive behavior, with the consequent increased risk of fouls during the game, is more likely to occur in individuals with high testosterone levels, not only in adulthood, but also during their intrauterine life.