Background: The number of H1N1 cases and death in India in recent years is good enough reason to declare it as a major emerging disease. AYUSH practitioners have been newly integrated into the National Health Delivery System. As they are the first level of contact for the community, their knowledge, attitude and right practices can help in counteracting the spread of H1N1. The objective of the study was to assess knowledge, attitude and practices regarding H1N1 among AYUSH practitioners at Panvel taluka.Methods: A cross sectional study was carried out during October-December 2015 among AYUSH practitioners practicing at Panvel taluka. A total of 225 AYUSH practitioners were included in the study by stratified probability proportional systematic random sampling. They were interviewed face to face by predesigned pretested questionnaire to assess knowledge, attitude and practices regarding H1N1. Attitude was measured on a four point Likert’s scale. Data was analysed by mean, percentage and Chi–square Test using software SPSS (version 21).Results: Only 32.4% had knowledge about period of infectivity by H1N1 virus. More than half (56.7%) knew incubation period of the disease.75% correctly answered on signs and symptoms of the disease. Only 25.3% knew the mode of administration of giving H1N1 live vaccine. 78.66% doctors strongly agreed that they can get infection and 82.66% agreed that they can be cured by the infection. Only 27.11% practitioners were vaccinated against H1N1. 43.12% doctors followed hand washing practice after seeing suspected case. 43.5% doctors used mask regularly at clinic. 59.6% doctors correctly prescribed drugs for prophylaxis and treatment of H1N1. 62.3% practitioners used to refer the patient in hospital designated by government for treatment of H1N1 cases.Conclusions: AYUSH practioners had average knowledge and poor practices about H1N1 prevention which can be further improved by continuing medical education programme.