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• Data on molecular epidemiology and antimicrobial resistance of Clostridium difficile are scarce. • The zoonotic potential of C. difficile is currently poorly understood. • In Zimbabwe a large strain diversity was present. • Low rates of antimicrobial resistance were observed (except for moxifloxacin). • Clusters of isolates from humans and chicken/soil could indicate for ongoing transmission. Clostridioides difficile is the major cause of infectious nosocomial diarrhoea in industrialized nations. Data on the occurrence of C. difficile in Africa, ribotype (RT) distribution, antimicrobial susceptibility patterns and potential zoonotic transmission are scarce. 80 Zimbabwean C. difficile isolates from different sources (chicken n = 30], soil n = 21] and humans n = 29]) were investigated using ribotyping, toxin gene detection, resistance testing, multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing (WGS). Among chicken isolates, the most common RTs were RT103 (6/30), RT025 (5/30) and RT070 (4/30). Within soil samples, RT025 and RT056 were most common (3/21 each). In contrast, the non-toxigenic RT084 was most frequently found in human isolates (4/29). Toxin genes were detected in only 19/29 human isolates. Susceptibility testing showed no resistance against metronidazole and vancomycin, and resistance against macrolides and rifampicin was scarce (3/80 and 2/80, respectively); however, 26/80 isolates showed moxifloxacin resistance. MLVA and WGS of strains with identical RTs stemming from different sources revealed clustering of RT025 and RT084 isolates from human und non-human samples. No "hypervirulent" strains were found. The detected clusters between human, chicken and soil isolates indicate ongoing transmission between humans and environmental sources and might point towards a zoonotic potential. [ABSTRACT FROM AUTHOR]