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Introduction: Little is known about prevailing ribotypes of Clostridium difficile infection in Romania where CDI is not a mandatory notifiable disease. Methodology: We studied 64 non-duplicate C. difficile isolates from patients hospitalised at the National Institute of Infectious Diseases, Bucharest, Romania between March 2011 and March 2012. Results: Sixty-three of the 64 C. difficile isolates produced toxins A and B whereas 44 (69%) isolates produced a binary toxin. Ribotype 027 accounted for 43 (68%) of the 63 toxigenic strains. The remaining 20 isolates belonged to ribotypes 018 (n = 9), 012 (n = 3), and, with one isolate each, 014, 031, 081, 416, 433, 500, 507 and PR03035 (new ribotype). Information on hospital mortality was available for 62 of the 64 patients; among these 62 cases, 4 (6.4%) ended fatal. Recurrence was documented for 11 (18.3%) of the 60 patients for whom this information was available. Multilocus variable-number tandem repeat analysis of the 43 isolates of ribotype 027 yielded a unique cluster for the Romanian isolates when compared to Austrian or Italian isolates. Conclusion: Our findings sustain the hypothesis of a recent emerged outbreak of C. difficile PCR ribotype 027 infections in the area of Bucharest. [ABSTRACT FROM AUTHOR]