Background. This cross-sectional study aimed to explore the microbial composition of the gut and its possible association with the Mediterranean diet (MD) after adjusting for demographic and anthropometric characteristics in a sample of healthy young Italian adults. Methods. Gut microbiota, demographic information, and data on adherence to MD and physical activity (PA) habits were collected in a sample of 140 university students (48.6% males, mean age 22.5 ± 2.9) with a mean body mass index (BMI) of 22.4 ± 2.8 kg/m2 (15.2–33.8) and a mean PA level of 3006.2 ± 2973.6 metabolic equivalent (MET)-minutes/week (148–21,090). Results. A high prevalence of Firmicutes and Bacteroidetes was found in all the fecal samples. Significant dissimilarities in the microbiota composition were found on the basis of MD adherence and PA levels (p = 0.001). At the genus level, Streptococcus and Dorea were highly abundant in overweight/obese individuals, Ruminococcus and Oscillospira in participants with lower adherence to MD, and Lachnobacterium in subjects with low levels of PA (p = 0.001). A significantly higher abundance of Paraprevotella was shown by individuals with lower BMI, lower MD adherence, and lower PA levels (p = 0.001). Conclusions. This study contributes to the characterization of the gut microbiome of healthy humans. The findings suggest the role of diet and PA in determining gut microbiota variability.