Introduction: Aerobic gram negative bacilli such as E. coli, Klebsiella pneumoniae and Enterobacter spp are major causes of hospital-acquired infections. Efflux pump mediated resistance is one of the major mechanism contributing to antimicrobial resistance in Enterobacterales. Aim/Objectives: The aim of our study was to detect efflux pump mediated resistance in Enterobacterales by Acridine orange agar method. Materials and Methods: In this study, various Enterobacterales were isolated from clinical samples and identified using various biochemical tests. The screening for expression of efflux pump was done by Acridine orange agar cartwheel method. Results: Among the Enterobacterales, E. coli showed highest prevalence of efflux pump activity followed by Klebsiella pneumoniae and Enterobacter spp. Conclusion: In this study, it is implicated that Acridine orange can be used as an alternative to Ethidium bromide and screening for efflux pump can be done by Acridine orange agar method in a microbiology laboratory set up.