Background Chemotherapy related mucosal toxicity is a major hindrance to successful therapy in pediatric cancers. The role of gut dysbiosis in modulation of chemotherapy related gastrointestinal toxicity is poorly understood. Methods Pediatric cancer patients with neutropenia and gastrointestinal symptoms were evaluated for neutropenic enterocolitis (NEC) with CECT abdomen. Clinical features, fecal calprotectin and microbiological data were analysed. Fecal Gut microbiota was evaluated in children with NEC and compared with children where NEC was excluded and healthy controls using conventional culture method. Results Of 590 children receiving chemotherapy during study period, 44 were diagnosed with NEC. Significantly higher frequency of isolation of Bacteroides was observed in children with NEC (42%) as compared to non- NEC group (14%) and healthy controls (13%). Isolation of Lactobacilli was infrequent in NEC group (26%) than non- NEC group (74%) and healthy controls (80%). There was nonsignificant trend towards higher isolation of Clostridium in children with NEC. Clostridiodes difficle or Clostridium septicum were not identified in any group. Isolation of other bacterial flora was similar in the sub groups. No significant association of survival with gut dysbiosis could be established. Isolation of Lactobacilli was associated with reduction in duration of intravenous alimentation by 2.4 days, whereas isolation of Bacteroides prolonged the requirement of bowel rest by 2.2 days. Conclusion Gut dysbiosis was significantly higher in NEC group and associated with higher morbidity suggesting its role in pathogenesis. This highlights role of interventions towards gut dysbiosis like prebiotics and probiotics in pediatric cancer patients.