© 2020 Bentham Science Publishers.Introduction: Topoisomerase II alpha (Topo IIα) has become one of the extensively exploited targets in chemotherapy due to its role in regulating the topological constraints of DNA during replication and transcription. Small molecules targeting Topo IIα’s activity such as etoposide (VP-16) and doxorubicin are extensively used in the treatment of many different types of cancer. Objective: Here, the effects of three small molecules, named as azacyanines, on Topo IIα have been assessed. Methods: In-vitro Topoisomerase IIα drug screening kit and agarose gel imaging were used for the assessment of Topo IIα’s activity. Results: Our results revealed that all the azacyanines investigated decreased the catalytic activity of Topo IIα dramatically. More importantly, the decrease in the catalytic activity of Topo IIα in the presence of azacyanines was higher than the presence of VP-16, which is a commercially available chemotherapy drug. Upon further investigation, it has been observed that Azamethyl’s catalytic inhibition of Topo IIα was concentration dependent and the catalytic activity of Topo IIα was almost completely abolished in the presence of 100.0 µM of Azamethyl. Conclusion: These findings reveal the potential of azacyanines as effective Topo IIα inhibitors and chemotherapeutic agents.