The bacterium originally named Bacillus difficilis was later renamed Clostridium difficile because of the difficulty associated with its isolation in the laboratory. C. difficile causes human-associated diarrhea, which is now known as C. difficile infection (CDI), a major cause of nosocomial infection mainly occurring in developed countries. Changes in antibiotic patterns in its strains produce toxins that are responsible for the high mortality rates associated with CDI; therefore, the epidemiology and severity of CDI have recently changed. Apart from CDI, C. difficile also causes opportunistic infections of the human gut usually when the normal gut flora are disrupted by broad-spectrum antibiotics. By disrupting normal gut flora, spores of C. difficile germinate and traverse the gut mucosa through flagellar binding to the mucosal epithelium where several proteins are involved in the binding of C. difficile. Proper diagnostic techniques have to be applied to ensure early identification of CDI and prompt treatment administered because false results may lead to inappropriate treatment and increase risk of cross-infection. This review discusses the epidemiology and pathogenicity of this bacterium with concern for its changing pattern over the years. Further details on the diagnosis of CDI are elaborated upon, mainly focusing on the limits of and challenges in molecular diagnosis.