Chronic stress may affect brain-gut axis and compromise blood-brain barrier (BBB) causing bacterial invasion and mounting nitrergic response that might be involved in stress-induced depression via direct impact on mitochondrial function in brain. The objectives of this study are to examine microbial composition in gut, blood and brain and assess a contribution of functionally different constitutive and inducible forms of nitric oxide synthase (cNOS and iNOS, respectively) to nitric oxide (NO) production in cytoplasm and mitochondria of cortico-limbic brain following chronic stress-induced depression. Depression-like behaviour of early adolescent male rats - resembling human depression - was developed by exposure to 2 weeks of chronic variable physical stress (CVS) (forced swimming, ether, restraint, cold, orthostatic shock and food deprivation).CVS induces in the rat gut a substantial enhancement the number of resident Candida albicans and manifestation of Staphylococcus aureus accompanied by a reduced number of obligate microbes immediately after CVS (stress group) and four days later (post-stress group). S. aureus and C. albicans were also detected in bloodstream and brain of CVS-treated rats. Simultaneously, the levels of L-arginine, L-citrulline and reactive nitrogen species were remarkably elevated in cytoplasm and mitochondria of the prefrontal cortex, striatum, hippocampus, and hypothalamus. We have found for the first time that CVS causes a persistent activation the iNOS in mitochondria of the mentioned brain regions, as well as in cytoplasm of the hypothalamus, while the cNOS activity was not significantly changed in mitochondria of the regions studied, with exception for the hypothalamic mitochondrial cNOS which reduced twice. We have also found a concurrent down-regulation the cNOS in cytoplasm of all the mentioned regions and a long-term up-regulation the iNOS in cytoplasm of the hypothalamus. CVS-induced opposite changes in the subcellular activity of distinct NOSs in cortico-limbic brain appear to be involved in the mitochondrial dysfunction leading to disturbances in neurotransmission, hemodynamic and energy impairment, pathophysiological pathways relevant to depression. We emphasize a role of gut microbiota in the persistent activation the mitochondrial iNOS in cortico-limbic brain and therefore the importance of origin the mitochondrial dysfunction involved in the pathological processes manifested during chronic stress-induced depression. This study would be helpful in understanding both brain-gut-microbiota communications and intracellular processes in the brain at depression, and therapeutic targeting of microbiome and nitrergic response might be included in future strategies in prevention and treatment of depression/anxiety