Background: It is becoming increasingly clear that the relationship between sleep disturbance and gut microbiota metabolites is of great importance. Aims: This study aimed to examine the changes in microbiota metabolites, brain-derived neurotrophic factors and synaptic proteins in insomnia patients, with a particular focus on the impact of diet. Methods: A total of 41 patients with insomnia and 45 healthy individuals participated in the study. The Food Frequency Questionnaire was employed to ascertain the subjects' daily macronutrient intake over the previous month. Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI) were used to evaluate insomnia complaints. The concentration of faecal short-chain fatty acids (SCFAs) was quantified by gas chromatography. Serum zonulin, brain derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95) and synaptophysin-like protein 1 (SYPL1) protein levels were quantified using an enzyme-linked immunosorbent assay (ELISA) method. Results: The total SCFAs, acetic acid, propionic acid, butyric acid and valeric acid levels were found to be significantly lower in the insomnia patient group compared to the control group. The levels of zonulin, PSD-95 and SYPL1 were found to be significantly elevated in the insomnia patient group in comparison to the control group. A significant negative correlation was observed between PSQI and ISI values and fatty acids. Conclusions: It has been demonstrated that sleep deprivation may be associated with alterations in the metabolites produced by the gut microbiota. In Western countries where dietary fibre consumption is low, increasing SCFA levels, promoting gut integrity and homeostasis may be regarded as a promising new approach for the treatment of diseases such as insomnia.