The influence of microbiota on the human body is currently the subject of many studies. The composition of bacteria colonizing the gastrointestinal tract varies depending on genetic make-up, lifestyle, use of antibiotics or the presence of diseases. The diet is also important in the species diversity of the microbiota. This study is an analysis of the relationships between physical activity, diet, and the microbiota of the gastrointestinal tract in athletes. This review shows the differences in the microbial composition in various sports disciplines, the influence of probiotics on the microbiome, the consequence of which may be achieved even better sports results. Physical activity increases the number of bacteria, mainly of the Clostridiales order and the genus: Lactobacillus, Prevotella, Bacteroides, and Veillonella, and their number varies depending on the sports discipline. These bacteria are present in athletes in sports that require a high VO2 max. The players’ diet also influences the composition of the microbiota. A diet rich in dietary fiber increases the amount of Lactobacillus or Bifidobacterium bacteria, probiotic microorganisms, which indicates the need to supplement the diet with probiotic preparations. It is impossible to suggest an unambiguous answer to how the microbiota of the gastrointestinal tract changes in athletes and requires further analyzes.