This study investigated the antibiotic susceptibility patterns and genetic resistance markers of 35 C. difficile strains isolated from patients with C. difficile infection. Vancomycin, metronidazole, tigecycline, teicoplanin, rifampicin, moxifloxacin, cefotaxime, tetracycline, erythromycin, clindamycin, chloramphenicol, linezolid and imipenem MICs were determined for toxigenic strains belonging to PCR ribotypes (PR) 012 (2), 014 (4), 017 (3), 018 (2), 027 (17), 046 (2), 087 (3) and 115 (2). Results showed vancomycin, metronidazole, tigecycline and teicoplanin to be active against all isolates. High resistance rates were noticed against cefotaxime (n = 35), clindamycin (n = 33), imipenem (n = 31), moxifloxacin (n = 25), erythromycin (n = 25) and rifampicin (n = 22). Linezolid-resistance was found in three isolates (PR 017/2, PR 012/1), showing complex resistance (7-9 antibiotics). PR 012, 017, 018, 027 and 046 isolates (n = 26) were resistant to 5-9 antibiotics. Twelve resistance profiles (2-9 antibiotics) were detected. Rifampicin-moxifloxacin-cefotaxime-erythromycin-clindamycin-imipenem-resistance was predominant, being expressed by 18 strains (PR 027/17, PR 018/1). PCR results suggested tetracycline-resistance to be induced by the gene tetM. Three tetM-positive isolates (PRs 012, 046), were also tndX-positive, suggesting the presence of a Tn5397-like element. Only two MLSB-resistant strains (PR 012) had the ermB gene and chloramphenicol-resistance determinant catD was not detected, leaving room for further investigating resistance mechanisms. Multidrug resistance could be attributed to most analysed strains, underlining, once more, the impact of wide-spectrum antimicrobial over prescription, still a tendency in our country, on transmission of antimicrobial resistance and emergence of epidemic C. difficile strains generating outbreaks.