Borrelia miyamotoi is a relapsing fever spirochete, first identified in Japan and later detected in various Ixodes species across the northern hemisphere. The pathogen was linked to human infections in North America, Europe, Asia, and Russia, causing symptoms ranging from flu-like illness to severe neurological damage. This study aimed to assess the presence of B. miyamotoi in Ixodes ricinus ticks collected from wildlife, dogs, and humans in Lombardy, Northern Italy, between 2020 and 2023. A total of 3,886 ticks were sampled, with a 1% positivity rate. The spirochete was detected in all life stages of ticks, with sequences 100% identical to European strains. The pathogen was found mostly in northern mountain regions. Although no human cases have been reported in Italy, these areas pose a potential risk due to high tourist activity.