This study aimed to evaluate the current status on the use of probiotics, disinfectants and antimicrobials in hatcheries, nurseries and grow-out farms producing Pangasius catfish (Pangasianodon hypophthalmus) in the Mekong Delta, Vietnam. Semi-structured interviews were conducted with 83 aquaculture enterprises (15 hatcheries, 32 nurseries and 36 grow-out farms). Farmers reported use of a total of 24 different antimicrobials, e.g. for treatment of bacillary necrosis and motile aeromonad septicaemia, and a variety of disinfectants, probiotics and nutritional supplements. In contrast to small-scale farmers, all large-scale grow-out farmers studied were certified and therefore had higher levels of formal education and specialized aquaculture training to diagnose and treat diseases. All farmers prepared their own medicated feed with a high risk of treatment failure, negative environmental impact from released antimicrobials and resistance development. Small-scale farmers were at particular occupational health risks when handling antimicrobials and other chemicals, e.g. mixing medicated feed with bare hands. There is an urgent need to improve knowledge and use innovative approaches, e.g. private-public partnerships, to assure a prudent use of chemicals, to improve capacity and access to disease diagnosis, particularly for small-scale grow-out farmers and nurseries. Efforts to control use of antimicrobials in aquaculture should be coordinated with the livestock and human health sectors taking an One-Health approach