In recent years Clostridium difficile infection (CDI) has represented a serious public health issue, mainly due to the global spread of the hypervirulent strain NAP1/027/BI. The purpose of the present study was to evaluate the utility of a PCR coupled with electrospray ionization mass spectrometry (ESI-MS) commercial assay for the detection of C. difficile virulence markers. Non-duplicative C. difficile isolates from patients with CDI diagnosed in a tertiary level hospital from Bucharest were tested for toxin A, toxin B, binary toxin genes and deletion in tcdC gene using PCR/capillary gel electrophoresis and PCR/ESI-MS. The study analysed 45 non-duplicative isolates, 33 strains (73.3%) belonging to ribotype 027. The concordance between PCR/capillary gel electrophoresis and PCR/ESI-MS was 100% for toxin A gene, 97.8% for toxin B gene, 91.1% for binary toxin subunit A gene and 95.6% for binary toxin subunit B gene. The general concordance for the complete panel of markers was 88.9% but was 100% for ribotype 027 isolates. PCR/ESI-MS might be a valid method for the detection of C. difficile virulence markers, including binary toxin.