•Adaptation of EtBr fluorescence assay for estimation of efflux pump activities.•The assay was effective in both Gram negative as well as Gram positive bacteria.•The spectrum of the assay was extended to diverse physiologies with efflux pumps.•The assay was used for first time directly on cells harvested from natural samples. Quick, simple and reliable assays to screen and understand active efflux pumps operative in pure bacterial cultures or mixed population in environmental samples are important. Although assays are available to monitor efflux activities in pure cultures, none has been used or tested directly on natural samples or cells harvested from them. Here, we have adapted an existing fluorescence assay using ethidium bromide (EtBr) to monitor efflux activities connected to resistance/tolerance to antibiotics (tetracycline), heavy metals (chromium), salt (NaCl) and ionic liquid (1-ethyl-3-methylimidazolium chloride) in different Gram positive and negative bacterial pure cultures. Concentration of 0.5 µg ml−1 of EtBr was found to be optimum for the assay. For all tested physiologies, 1.2–2.2 fold lower EtBr fluorescence (or higher efflux) was detected as compared to control. In addition, the assay was efficiently used for the first time to establish differences in efflux activities in microbial cells harvested from different natural environments. Similar to pure cultures, sediments with higher salinity (7%) compared to lower (3%) ones, and antibiotic contaminated hospital drain samples compared to adjoining garden soil showed up to 1.7-fold and 1.3-fold higher efflux activities respectively. Thus, EtBr fluorescence assay can be used effectively in known or novel bacterial physiologies that involve efflux activities not only in pure cultures, but also in natural environmental microbial groups.