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Leptospirosis is a neglected zoonotic disease that is endemic in tropical regions, including Ecuador. It is caused by spirochetes of the genus Leptospira, which can infect humans through animal reservoirs such as rats and dogs, or through contact with contaminated water or soil. In March 2023, public health authorities declared a concerning outbreak of leptospirosis in Durán Cantón, located in the Coastal region of Ecuador. For the first time in the country, a multidisciplinary approach involving physicians and veterinarians was implemented for the surveillance and management of this leptospirosis outbreak. A total of 335 samples were collected, including suspected human cases, household contacts, household dogs, synanthropic rats, and water samples within the area of human cases. Samples were processed by qPCR targeting lipL32, secY, and rrs fragment genes and characterized further for Sanger sequencing. Overall, 26.2 % of human samples, 43.8 % of dog samples, 38.5 % of rat samples, and 39.4 % of water samples tested positive for Leptospira. Further, phylogenetic analysis shows that human, dog, and rat sequences are clustered within the pathogenic subclade P1, within the branch of L. kirschneri and L. interrogans. This study is the first of its kind in Ecuador, where an ongoing outbreak of leptospirosis was managed in real-time by using molecular diagnosis and not serological tools, and where the epidemiological surveillance was done following a One Health approach. This experience should inspire public and animal health authorities in Ecuador to promote a national One Health surveillance and control program for zoonotic diseases.