Background Studies assessing consumers’ knowledge of the rational use of antibiotics are essential to understand the knowledge gap before intervention strategies are instituted. Objectives To assess the knowledge of rational use of antibiotics among consumers in Dar es Salaam, Tanzania. Methods A cross-sectional study assessing knowledge of rational use of antibiotics among 960 consumers was conducted in Dar es salaam in March 2021. Participants were consecutively enrolled from outpatient pharmacies in selected public and private hospitals and marketplaces in Ilala Municipality. Data were collected using the WHO-validated questions on knowledge of consumers of antibiotic uses. Results Overall, 196 (20.4%) and 503 (52.4%) participants demonstrated good knowledge of rational antibiotic use and conditions that can be treated with antibiotics, respectively. However, 678 (70.6%) responded that they stopped using antibiotics after dose completion, 515 (53.6%) would request the same antibiotic if it had helped to treat a similar condition in the past and 406 (42.3%) are willing to use the same antibiotic if a friend or family member used the medication previously to treat similar signs and symptoms. Besides, the following conditions were mentioned as being treatable with antibiotics: influenza (50.7%), sore throat (61.4%) and urinary tract infection (60.5%). Conclusions The majority of the consumers had poor knowledge of the rational uses of antibiotics and a moderate proportion had good knowledge of the conditions that are treatable with antibiotics. Those with a high level of education and with health insurance had good knowledge of rational uses of antibiotics.