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Introduction: Co-infection of influenza A and B has been reported, especially in outbreak situations, but epidemiological and clinical information is limited. We aimed to investigate an outbreak of influenza among health care workers in which the index case suffered from influenza A and B co-infection. Methodology: We investigated the outbreak setting through the utilization of structural questionnaires, molecular methods, and serological tests. Results: Among 13 persons, one index case and five confirmed secondary cases were confirmed. The overall influenza infection rate was 46.2% (6/13), with infection rates for influenza A and B at 38.5% (5/13) and 23.1% (3/13), respectively. Interestingly, one of the secondary cases had influenza A and B co-infection identical to the index case. There was no significant association between vaccination status and influenza infection. Conclusions: This study unveils the demonstration of human-to-human influenza A and B co-infection transmission for the first time. Surveillance systems, combined with epidemiological case investigation comprising molecular diagnosis, should be strengthened for future influenza outbreak preparedness. [ABSTRACT FROM AUTHOR]