Although the gut microbiota is known to affect body weight, its relationship with overweight/obesity is unclear. Our aim was to characterize microbiota composition in a cohort from the southernmost area of Italy. We investigated whether an altered gut microbiota could play an etiological role in the pathogenesis of overweight/obesity. A total of 163 healthy adults were enrolled. Microbiome analysis was performed via 16S rRNA gene sequencing. We found significant phylum variations between overweight (N = 88) and normal-weight (N = 75) subjects.BacteroidetesandProteobacteriawere higher in overweight participants (p= 0.004;p= 0.03), andFirmicutesandVerrucomicrobiawere lower (p= 0.02;p= 0.008) compared to normal-weight participants. Additionally,AkkermansiaandBifidobacterium(genus level) were significantly lower in the overweight group, as well asAkkermansia muciniphilaat the species level. TheFirmicutes/Bacteroidetesratio (F/B ratio), an index of dysbiosis, was found to be inversely associated with BMI in linear and logistic regression models (p= 0.001;p= 0.005). The association remained statistically significant after adjustment for potential confounders. This cross-sectional study contributes to defining the gut microbiota composition in an adult population living in southern Italy. It confirms the relationship between overweight susceptibility and the dysbiosis status, highlighting the possible etiological role of the F/B ratio in disease susceptibility.