Aquaculture plays an important role in global food supply and in national economic development in low and middle income countries. However, during the process of aquaculture production, farmer may inappropriately use antibiotics to prevent and to treat infections. This may lead to antimicrobial resistance in humans and environment. This study aimed to assess fisheries officers’ knowledge and awareness of antibiotic use, antimicrobial resistance, and knowledge on regulations related to antibiotic use in aquaculture sector. A self-administered questionnaire survey was conducted in 92 fisheries officers whose work was related to aquatic animal health and aquaculture management from 61 provinces in February 2019. The questionnaire comprised four parts: 1) knowledge about the Thailand’s National Strategic Plan on Antimicrobial Resistance 2017-2021 (NSP-AMR); 2) knowledge on antibiotics; 3) awareness of antimicrobial resistance; and 4) regulations on chemicals and antibiotic use in aquatic animals. Over half of the respondents (57.6%) had heard about the NSP-AMR, but most of them (62.3%) did not know the content. The level of knowledge of antimicrobial was measured using six true/false statements. Results showed that level of knowledge on AMR and antibiotic use was high, as 73.9% of respondents gave correct answers to more than 3 out of 6. The majority of respondents (more than 80.0%) correctly recognized the importance of antibiotic use and AMR problems, and all of the respondents agreed that AMR was an important problem that needed policy attention. More than half of respondents gave correct answers in all statements regarding the antimicrobial use regulations in aquaculture sector. This study was the first study which assessed knowledge on antibiotic use and antimicrobial resistance among fisheries officers in Thailand. We applied the questionnaire from the National Health and Welfare Survey 2019 and the results showed that respondents had a high level of knowledge about antibiotic use and antimicrobial resistance. But, gaps remain in the dissemination of antibiotic-related policies. Training should be provided to improve knowledge and awareness on AMR and to support fisheries officers in regulating and ensuring rational use of antibiotics in the aquaculture.