Behavioral dysfunctions in dogs represent one of the main social concerns, since they can endanger both humans and animals. Among them, phobia is usually characterised by a strong and persistent fear towards something that might feel dogs threatening, including thunderstorms, fireworks, strangers, or sounds. Alongside with genetic susceptibility, environmental factors, traumatic experiences, and medical conditions can cause dog to develop such psychiatric disorders. The available treatments rely on behavioral therapy, based on cognitive-relational approach, associated to the administration of drugs acting on GABA, serotonin and catecholamines. Unfortunately, pharmacological interventions are hardly effective and discourage the owners, who often decide to relinquish the animals to the shelters. Recent and growing evidence in humans and animals highlight the importance of the gut-brain axis in the modulation of the brain physiology and behavior. Therefore, in the present study we sought to investigate the potential connection between gut microbiota and phobia in companion dogs. To this aim, we first analyzed the faecal microbiota in samples (total n = 16) collected from mixed breed adult dogs (n = 8/group). Differentially abundance analysis allowed us to identify taxa related to Proteobacteria and Firmicutes Phyla, significantly altered in phobic dogs. In addition, metabolome analysis on the blood serum samples of the same phobic dogs showed significant alterations of molecules associated to GABA and glutamate neurotransmission, as well as bile acids metabolism. Collectively, our preliminary data may shed light on the importance of combining behavioral and high-throughput approaches to improve diagnosis and therapy, aimed at counteracting phobia in dogs.