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The consumption of an optimal amount of fruits and vegetables is known to improve physical fitness and physiological body functions. Healthy eating habits, including intake of fruits and vegetables, can modify gut microbiota. This study aimed to demonstrate the effectiveness of a formulated fruit and vegetable supplement (FVS) in modulating the antioxidant capacity and the gut microbiota composition. We enrolled 30 healthy volunteer subjects, matched for age, gender, BMI, and smoking habits, and randomized them into the FVS and the placebo (PLA) groups. Among the serum vitamins, the folic acid level was significantly higher (<i>p</i> = 0.001) in the FVS group than in the PLA group, whereas the vitamin B2 level was significantly higher in the PLA group than in the FVS group (<i>p</i> = 0.028). The antioxidant capacity, measured by using the oxygen radical absorbance capacity (ORAC) method, was also slightly higher in the FVS group than in the PLA group but did not reach statistical significance. The dietary intake, assessed by 24-h recalls, did not show any significant changes after the supplementation in both the groups. The gut microbiome composition, measured by 16S rDNA sequencing, showed no difference in both alpha and beta diversities, whereas the LEfse analysis revealed a microbial shift after the treatment, with a decreased abundance of the genus <i>Ruminococcus</i> from the Lachnospiraceae family (<i>p</i> = 0.009), and the unclassified genus from the family Erysipelotrichaceae (UC36, <i>p</i> = 0.003) in the FVS group compared with the PLA group (confirmed by SIAMCAT analysis, AUC = 74.1%). With a minor effect, the genus <i>Faecalibacterium</i> and unclassified genus and family from the order Lactobacillales (UC31) were also increased in the FVS group compared with the PLA group (<i>p</i> = 0.0474, <i>p</i> = 0.0352, respectively). SCFA measurement by gas chromatography-mass spectrometry showed an increased level of 2-methylbutyrate in the FVS group compared with the PLA group (<i>p</i> = 0.0385). Finally, the Spearman correlation analysis showed that in the FVS group, the genus <i>Faecalibacterium</i> positively correlated with 2-methyl butyrate (<i>p</i> = 0.040). In the PLA group, none of the significant bacteria correlated with either SCFA or serum biomarkers. The network analysis confirmed the positive correlation between genus <i>Faecalibacterium</i> and 2-methyl butyrate. We can conclude that the FVS in healthy individuals modified the gut microbiota composition and metabolites, and it can potentially contribute to reduce the pro-inflammatory response along with the antioxidant capacity.