Antibiotics strongly disrupt the human gut microbiota, which in consequence loses its colonization resistance capacity, allowing infection by opportunistic pathogens such as Clostridium difficile. This bacterium is the main cause of antibiotic-associated diarrhea and a current problem in developed countries, since its incidence and severity have increased during the last years. Furthermore, the emergence of antibiotic resistance strains has reduced the efficiency of the standard treatment with antibiotics, leading to a higher rate of relapses. Here, we review recent efforts focused on the impact of antibiotics in the gut microbiome and their relationship with C. difficile colonization, as well as, in the identification of bacteria and mechanisms involved in the protection against C. difficile infection. Since a healthy gut microbiota is able to avoid pathogen colonization, restoration of the gut microbiota seems to be the most promising approach to face C. difficile infection, especially for recurrent cases. Therefore, it would be possible to design probiotics for patients undergoing antimicrobial therapies in order to prevent or fight the expansion of the pathogen in the gut ecosystem.