Obesity is a common metabolic disorder caused by a sedentary lifestyle, and a high-fat and a high-glucose diet in the form of fast foods. High-fat diet-induced obesity is a major cause of diabetes and cardiovascular diseases, whereas exercise and physical activity can ameliorate these disorders. Moreover, exercise and the gut microbiota are known to be interconnected, since exercise can increase the gut microbial diversity and contribute to the beneficial health effects. In this context, we analyzed the effect of diet and exercise on the gut microbiota of mice, by next-generation sequencing of the bacterial V4 region of 16S rRNA. Briefly, mice were divided into four groups: chow-diet (CD), high-fat diet (HFD), high-fat diet + exercise (HFX), and exercise-only (EX). The mice underwent treadmill exercise and diet intervention for 8 weeks, followed by the collection of their feces and DNA extraction for sequencing. The data were analyzed using the QIIME 2 bioinformatics platform and R software to assess their gut microbial composition, richness, and diversity. The Bacteroidetes to Firmicutes ratio was found to be decreased manifold in the HFD and HFX groups compared to the CD and EX groups. The gut microbial richness was comparatively lower in the HFD and HFX groups and higher in the CD and EX groups (ACE, Chao1, and observed OTUs). However, the Shannon alpha diversity index was higher in the HFD and HFX groups than in the CD and EX groups. The beta diversity based on Jaccard, Bray–Curtis, and weighted UniFrac distance metrics was significant among the groups, as measured by PERMANOVA. Paraprevotella, Desulfovibrio, and Lactococcus were the differentially abundant/present genera based on the intervention groups and in addition to these three bacteria, Butyricimonas and Desulfovibrio C21c20 were differentially abundant/present based on diet. Hence, diet significantly contributed to the majority of the changes in the gut microbiota.