Aims and Objectives: Intracanal medication plays an important role in disinfecting the root canal from various microorganisms. There are various intracanal medication frequently used by practitioners based on the efficacy of the same, therefore to compare antimicrobial activity of a triple antibiotic paste with chlorhexidine gel and calcium hydroxide against microorganisms in the root canal used as irrigating solution. Materials and Methods: The present study was conducted on 88 single rooted (central and lateral incisors), asymptomatic, nonvital, and necrotic teeth. Individuals included were between the ages of 12–15 years with no systemic complications. Overall, 88 teeth were divided randomly into four groups on the basis of treatment they were given, with each group having 22 teeth. Each group was assigned to one intervention group namely 2% chlorhexidine gel group, calcium hydroxide group, triantibiotic paste group, and the normal saline group. Triantibiotic paste was prepared from ciprofloxacin Hydrochloride 500 mg+ Metronidazole 400 mg+ Tetracycline 500 mg. Two samples were collected from individual tooth to assess the change in the extent of total colony forming units. Results: Mean log 10 bacterial count among the triantibiotic paste was 5.222 preirrigation which was decreased to 0.653. Maximum percentage of log 10 bacterial count was decreased among triantibiotic paste group of 87.20% while control group of normal saline has recorded a decrease of 54.65% in bacterial count. Among all the four groups, there was a decrease of 73.51% of bacterial count. Conclusion: From the above results, it was concluded that triantibiotic paste demonstrated the highest efficacy against bacterial pathogen when used as irrigating solution.