This prospective observational cohort study aimed to establish and compare baseline rates of Clostridioides difficile infection (CDI) in community and hospitalized patients in Nagpur and rural Melghat Maharashtra, including adults aged ≥18 years with a diagnosis of diarrhoea as defined as 3 or more loose stools in a 24 h period. All diarrhoeal samples were tested for CDI using the C. diff Quik Chek Complete enzyme immunoassay. C. difficile-positive stool samples were characterised by toxigenic culture, antimicrobial susceptibility testing and PCR ribotyping. C. difficile testing was performed on 1683 patients with acute diarrhoea. A total of 54 patients (3.21%; 95% CI: 2.42–4.17) tested positive for both the GDH antigen and free toxin. The risk factors for CDI included the presence of co-morbidities, antibiotic usage, and immunosuppression. The detected PCR ribotypes included 053-16, 017, 313, 001, 107, and 216. Our findings show that toxigenic C. difficile is an important but neglected aetiologic agent of infective diarrhoea in Central India. These results underscore the need to enhance the awareness and testing of patients with diarrhoea in India regarding the presence of toxigenic C. difficile, particularly in high-risk individuals with multiple co-morbidities, immunosuppression, and recent or ongoing antibiotic exposure or hospitalization.