Introduction: Multidrug-resistant (MDR) strains of bacteria pose a major threat in clinical settings. Multidrug resistance can be due to various mechanisms but is primarily the result of over-expressed efflux pumps that extrude unrelated antibiotics before they reach the intended targets. The emergence of MDR due to efflux pumps has to lead to a diagnostic and therapeutic dilemma. Ethidium Bromide (EtBr)-agar cartwheel assay is a newly discovered simple, safe and cost-effective method to determine efflux pump activity. Objectives: The study aimed at the detection of efflux pump activity in MDR strains belonging to Enterobacteriaceae family using the EtBr cartwheel method as well as determining the antimicrobial susceptibility pattern of MDR Enterobacteriaceae. Materials and methods: A total of 95 MDR Enterobacteriaceae isolates from various clinical samples were included in the study. Identification and antimicrobial susceptibility were done following Clinical Laboratory Standards Institute(CLSI) 2019 guidelines. They were evaluated for efflux activity using the EtBr agar cartwheel method. Data analysis was performed using SPSS version 20. Results: All the 95 isolates were tested for efflux pump using the Ethidium Bromide agar cartwheel technique. The results showed that 47(49.47%) of the total isolates gave positive results. Among the 47 efflux pump, positive organisms majority were Klebsiellapneumoniae, followed by Escherichia coli and Proteus mirabilis. Conclusion: Laboratory detection of efflux pumps in bacteria can be effectively done using EtBr agar cartwheel assay. We conclude that over-expression of efflux pumps has led to an alarming rise in drug resistance and necessary steps should be taken to control this problem.