Childhood obesity continues to represent a growing challenge, and it has been associated with gut microbiota dysbiosis. This study examines the gut microbiota composition in overweight and obese school children and assesses whether a 12-week multidisciplinary intervention can induce changes in the gut microbiota. The intervention, which combined recreational football and nutritional education, was implemented among 15 school children, aged 7-10 years, with a Body Mass Index ≥ 85th percentile. The children were assigned into two groups: Football Group ( = 9) and Nutrition and Football Group ( = 6). Faecal samples were collected at the beginning and end of the program and analysed by sequencing the 16S rRNA gene. Over the intervention, a significant decrease was found collectively for Bifidobacterium genera ( = 0.011) and for genera in the Football Group ( = 0.021). The relative abundance of ( = 0.002) and ( = 0.009) was negatively correlated with moderate to vigorous physical activity (MVPA), while was positively correlated with MVPA ( = 0.010) and with the daily intake of protein ( = 0.008). Our findings suggest that a multidisciplinary intervention was capable of inducing limited but significant positive changes in the gut microbiota composition in overweight and obese school children.