The gut microbiota is currently recognized as an important factor influencing the host’s metabolism, immune, and central nervous systems. Determination of the composition of the gut microbiota of healthy subjects is therefore necessary to establish a baseline for the detection of alterations in the microbiota under pathological conditions. So far, most studies describing the gut microbiota have been performed in populations from Asia, North America, and Europe, whereas populations from Africa have been overlooked. Here, we present the first characterization of the intestinal microbiota in healthy Tunisian adults using 16S rRNA gene sequencing. We further compare the gut microbiota composition based on gender and BMI. Our results showed that the Tunisian gut microbiota is dominated by the phyla Firmicutes and Bacteroidota in accordance with studies from western countries. However, some specificities have been identified, including a higher proportion of Firmicutes in males and higher proportions of Atopobiaceae and Peptostreptococcaceae in Tunisian overweight individuals. Moreover, we were able to identify bacterial species differently represented between males and females and between normal weight and overweight individuals. These results constitute an important baseline that can be used to identify the dysbiosis associated with the main diseases affecting the Tunisian population.