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Abstract Background C. difficile has been increasingly reported as a cause of gastrointestinal disease in children, ranging from mild self-limiting diarrhea to severe conditions such as pseudomembranous colitis and toxic megacolon. Only two pediatric research groups reported the presence of C. difficile infection in Brazilian children, but no previous research has examined C. difficile infection among children in northeastern Brazil. This prospective cross-sectional study investigated the molecular epidemiology and antimicrobial resistance of C. difficile strains isolated from children and adolescents with diarrhea referred to a tertiary pediatric hospital in Brazil while exploring the associated risk factors. Results Toxin positivity or C. difficile isolation was found in 30.4 % (17/56) samples. C. difficile was isolated from 35 % (6/17) samples. Four toxigenic strains were identified (tpi+, tcdA+, tcdB+, cdtB-, without tcdC deletions) belonging to PCR ribotypes and PFGE-pulsotypes: 046 (new pulsotype 1174), 106 (NAP11), 002 (new pulsotype 1274), 012 (new pulsotype NML-1235). Two of the six isolates belonging to ribotypes 143 and 133 were non-toxigenic. All toxigenic strains were sensitive to metronidazole and vancomycin. Regarding the clinical manifestation, diarrhea lasted an average of 11 days, ranging from 3 to 50 days and was often associated with mucus and/or blood. All six patients from whom the C. difficile was isolated had a chronic disease diagnosis, with these comorbidities as the main risk factors. Conclusion Our study enhances our understanding of the present epidemiological landscape of C. difficile-associated diarrhea (CDI) among children in northeastern Brazil, reveling a substantial CDI frequency of 30.4 %, with toxigenic strains detected in 76.4 % of cases, highlighting a higher prevalence compared to earlier Brazilian studies. In the globalized world, an understanding of disease-generating strains, the associated risk factors, clinical manifestation, and antimicrobial sensitivity has fundamental epidemiological importance and draws attention to preventive measures, allowing for more decisive action. C selflimiting self limiting megacolon crosssectional cross sectional factors 304 30 4 30. 17/56 1756 17 56 (17/56 samples 6/17 617 6 (6/17 tpi+, tpi (tpi+ tcdA tcdA+ tcdB tcdB+ cdtB, cdtB , cdtB- deletions PFGEpulsotypes PFGE pulsotypes PFGE-pulsotypes 04 new 1174, 1174 1174) 10 NAP11, NAP11 NAP (NAP11) 00 1274, 1274 1274) 01 NML1235. NML1235 NML 1235 . NML-1235) 14 13 nontoxigenic. nontoxigenic non toxigenic. non-toxigenic vancomycin manifestation 1 5 andor blood diagnosis difficileassociated (CDI 764 76 76. cases studies world diseasegenerating generating measures action 17/5 175 (17/5 6/1 61 (6/1 tpi+ (tpi 0 117 NAP1 (NAP11 127 NML123 123 NML-1235 7 17/ (17/ 6/ (6/ (NAP1 12 NML12 NML-123 (17 (6 (NAP NML1 NML-12 (1 ( NML-1 NML-