Introduction: Anxiety and stress are prevalent mental health issues. Traditional drug treatments often come with unwanted side effects and may not produce the desired results. As an alternative, probiotics are being used as a treatment option due to their lack of specific side effects. Probiotics are live microorganisms that, in sufficient amounts, benefit the host’s health. The human digestive tract naturally contains hundreds of different types of bacteria; among these bacteria, probiotic strains of Lactobacillus rhamnosus DM163 and Lactobacillus delbrueckii DPUL-F36 are among the species that may improve behavioral disorders such as anxiety and stress. Methods: In this study, 42 adult male Wistar rats (190–220 gr/BW), categorized into six groups, were used. Except for the control group, the rest were subjected to 30 days of light–dark cycle alteration (4 h dark, 20 h light). Simultaneously with the light/dark disruption, these groups were gavaged daily doses of 1 × 10 9 colony-forming units (CFU) of the target probiotics. Stool samples were collected to confirm changes in the gut microbiota before and after administration of probiotics. Following DNA extraction, PCR was performed using specific primers. The open-field test and the elevated plus maze test were used to check the anxiety, stress, and exploratory behaviors. Results: The comparison of behavioral tests showed that the change in the light–dark cycle caused negative behavioral changes, and the administration of probiotics, particularly L. rhamnosus, was found to be more effective in reducing anxiety and stress levels and improving exploratory behavior compared to L. delbrueckii. The qualitative PCR test also determined that during the 30-day intervention period, L. rhamnosus and L. delbrueckii bacteria were present in the intestinal bacterial flora of rats. Discussion: Changes in the light–dark cycle cause significant disturbances in normal physiology. These alterations are especially evident in the functions of the central nervous system and various behaviors. So, in the long term, it can seriously lead to destructive neurodegenerative alterations of the nervous system. Probably, the increase and predominance of the population of probiotics and the effects of their metabolites using the gut–brain axis will lead to beneficial and even preventive effects.

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