The relationship between the gut microbiota and chronic insomnia remains understudied. The aim of this paper was to investigate changes in the taxonomic composition of the gut microbiota and their associations with the levels of cortisol, melatonin and IL6 in patients with chronic insomnia. Our comparative prospective cross-sectional study enrolled 55 patients with chronic insomnia, who formed the main group (female patients: 58.2%, male patients: 41.8%; mean age 31.6 ± 7.4 years), and 50 healthy volunteers, who comprised the control group (females: 68.0%, males: 32.0%; mean age 33.2 ± 6.6 years). The taxonomic composition of the gut microbiota was profiled using 16S rRNA gene sequencing. Plasma cortisol and IL 6 and urine melatonin were measured by means of ELISA. Sleep quality was evaluated using the Pittsburgh Sleep Quality Index (PSQI). In patients with chronic insomnia, the abundance of Faecalibacterium (p = 0.048), Prevotella 9 (p < 0.001) and Lachnospira (p = 0.036) was lower, whereas the abundance of Blautia (p = 0.012) and Eubacteriumhallii (p = 0.003) was higher than in healthy volunteers. Significant correlations were established between the levels of IL6 and the abundance of Faecalibacterium (r = –0.44; p = 0.001) and Blautia (r = 0.42; p < 0.001), as well as between cortisol concentrations and the abundance of Lachnospira (r = –0.41; p = 0.048). The abundance of Faecalibacterium and Blautiaс was correlated with higher PSQI (r = –0.47, p = 0.001; r = 0.45, p < 0.001, respectively). Our study contributed to the pool of data about changes in the gut microbiota and their associations with some endocrine and inflammation markers in patients with chronic insomnia. These data can be exploited to propose new strategies for the diagnosis and personalized treatment of insomnia aimed at normalizing the patient’s gut microbiota.