Aim: To assess the knowledge and practices regarding antibiotic prescription and development of resistance among private dental practitioners in Chennai city, Tamilnadu, India. Method: A cross-sectional survey was conducted among 280 practitioners in Chennai registered under IDA, selected by simple random sampling. A structured proforma was administered, to obtain data. Descriptive statistics were calculated and Chi-square test was carried out to check the significant difference. Level of significance was set at 5%. Results: Majority of the participants were specialists (62.1%). Study participants with postgraduate qualification were more likely to know the current guidelines for antibiotic prophylaxis and was found to be highly statistically significant (p=0.000) Many practitioners believed that self-prescription by patient (84.3%) and over-prescription by dentist (74.3%) may lead to the development of AMR. Conclusion: The present study results concluded that low knowledge and guideline-incongruent therapeutic prescribing practices were observed. This indicates a need for development of targeted interventions to promote judicious antibiotic use among the dentists.