The aim of the present study was to evaluate the effect of cystic fibrosis and antibiotic therapy on intestinal microbiota composition and intestinal inflammation in children and adolescents. A cross-sectional controlled study was conducted with 36 children and adolescents: 19 in the cystic fibrosis group (CFG) and 17 in the control group (CG) matched for age and sex. The CFG was subdivided based on the use of antibiotic therapy (CFAB group) and non-use of antibiotic therapy (CFnAB group). The following data were evaluated: colonization, antibiotic therapy, mutation, breastfeeding, use of infant formula, type of delivery, introduction of solid foods, body mass index, fecal calprotectin and intestinal microbiota composition (fluorescence in situ hybridization). Intestinal inflammation evaluated by fecal calprotectin was significantly higher in the CFG (median: 40.80 µg/g, IQR: 19.80-87.10, p = 0.040) and CFAB group (median: 62.95 µg/g, IQR: 21.80-136.62, p = 0.045) compared to the CG (median: 20.15 µg/g, IQR: 16.20-31.00), and the Bacteroides, Firmicutes, Eubacterium rectale and Faecalibacterium prausnitzii were significantly decreased (p < 0.05) in the CFG compared to the CG, whereas the bacteria Clostridium difficile, Escherichia coli and Pseudomonas aeruginosa were significantly increased in the CFG (p < 0.05). The main differences were found between the CG and CFAB group for Eubacterium rectale (p = 0.006), Bifidobacterium (p = 0.017), Escherichia coli (p = 0.030), Firmicutes (p = 0.002), Pseudomonas aeruginosa (p < 0.001) and Clostridium difficile (p = 0.006). The results of this study confirm intestinal inflammation in patients with CF, which may be related to changes in the composition of the intestinal microbiota.