Introduction Inappropriate use of antibiotics contributes significantly to the development of antimicrobial resistance. Weak or lack of drug regulation and a lack of veterinarian supervision during antibiotic administration in LMICs, result in the indiscriminate use of antibiotics for therapeutic or growth promotion purposes in aquaculture, livestock and agriculture. Consequently, the deposition of antimicrobial residues in the food chain may result, posing a risk to food safety and accelerating the development of Antimicrobial Resistance (AMR). Of particular concern is the use of critically important antibiotics for human health, as prophylaxis in animals (Brunton, L. A et al., 2019). This study assesses the Knowledge, Attitudes and Practices surrounding safety and use of antimicrobials amongst aquaculture farmers within Nairobi County, Kenya. Methods A prospective list of 24 aquaculture farmers was provided by the Nairobi County Fisheries office and reduced to 21, as three farmers had withdrawn from aquaculture farming. A further three farmers were unavailable to participate, leaving 18 farmers for inclusion in this study. A semi-structured questionnaire was administered using Kobo Toolbox from ODK-collect. Farmers were surveyed on Knowledge, Attitude and Practice (KAP) questions relating to prudent use of antibiotics in aquaculture. Data cleaning and processing were performed in Microsoft Excel and STATA (Stata/IC 16.1 for Mac) . Responses were scored to create composite KAP variables for univariate analysis by Chi2 test. Results Only two out of the 18 farmers were reported to be using antibiotics (Tetracyclines and colistin in combination) in their aquaculture farms. Generally, the study showed a poor understanding of the terms ‘antibiotics’ and ‘antimicrobial resistance’ amongst the farmers, and limited recognition of fish diseases or knowledge on when to apply antibiotics. While 88.89% reported having sufficient knowledge on antibiotic use, and 72.22% a favourable attitude towards prudent antibiotic use, this study found 66.67% demonstrated inadequate practice around antibiotic use. Conclusions This study suggests low reported usage of antibiotics, which does not equate to prudent use of antibiotics in aquaculture farming within Nairobi County, Kenya. There have been national awareness raising campaigns in Kenya, which may explain findings in this study showing some knowledge of AMR and favourable attitudes to prudent use of antibiotics, However, the Ministry of Livestock and Agriculture, Department of Fisheries, and the Directorate of Veterinary Services (DVS) need to strengthen sensitization amongst farmers on fish health management, antibiotic stewardship leadership and access to appropriate antimicrobials for aquaculture. Kenya Veterinary Medicines Directorate (VMD) needs to authorize specific antibiotics for use in aquaculture farming to reduce or prevent usage of critically important antibiotics for human medicine, such as colistin in aquaculture.