Purpose This study aimed to assess the knowledge, attitude, and practice among mothers on antibiotic use in under-5 children with respiratory tract infection attending the pediatric outpatient department at Sri Ramachandra Hospital in Chennai city, South India. Research design A cross-sectional descriptive design was adopted in this study. Methods The target population was mothers of under-5 children attending the pediatric outpatient department in the G block of Sri Ramachandra Hospital, Chennai city, South India. The sample size of 240 was estimated by a single proportion. The non-probability convenience sampling technique was used to select samples. A structured questionnaire developed by the investigator based on previous study literatures was used to assess the knowledge, attitude, and practice of antibiotic use in respiratory tract infections. Results The study revealed that 49.6% of mothers had moderately adequate knowledge, 92.1% of mothers had moderately favorable attitude, and 70% of mothers had unsatisfactory practice. There was a statistically considerable positive correlation between knowledge and attitude (p < 0.05) and attitude and practice (p < 0.001). There was a significant association between knowledge on antibiotic use and number of children, education, occupation, and monthly family income of mothers at a significant level of p < 0.05 and p < 0.01, respectively. There was a significant association between attitude on antibiotic use and age of child at p < 0.05. There was a significant association between practice on antibiotic use and education, age of child at levels of p < 0.05 and p < 0.01, respectively. Conclusions This study suggested that perfection in the appropriate use of antibiotics would require multi-sectorial cooperation and long-term efforts. Healthy communication with mothers through mass media will be useful in enhancing and promoting changes in mothers' knowledge, attitude, and practice towards the use of antibiotics for their children, especially among parents from rural areas and with no formal education.