Abstract Aim: To evaluate modifications of gut microbiota after antibiotic therapy in breast-fed infants. Study design: Twenty-six exclusively breast-fed infants younger than 5 months hospitalized for pneumonia treated with ceftriaxone (50 mg per kilo per day administered intramuscularly) were recruited. Intestinal microbiota at day 0 – before starting antibiotic administration – at the end of the therapy (5 days after) and after 15 days after the stop was analysed. Stool samples were collected and immediately diluted and cultured on selective media to detect total bacteria, Enterobacteriaceae, enterococci and lactobacilli. Statistical analysis was performed by using Wilcoxon test. Results: After 5 days of antibiotic therapy, we observed a significant reduction in total faecal bacterial count (p=0.003) in Enterobacteriaceae (p=0.001) and enterococci (p<0.001), in comparison with day 0. After 5 days of therapy, lactobacilli are no longer detected. Conversely, bacterial count values for all bacteria detected after 15 days from the end of therapy are significantly increased and similar to day 0. Conclusion: Our findings showed that gut microbiota was significantly modified after 5 days of antibiotic therapy; exclusively, breast-feeding may be relevant in promoting the re-establishment of gut microbiota composition in early infancy.