Waterborne diseases cause a significant health burden worldwide because of water contamination. According to the World Health Organization (WHO), in 2020, worldwide, approximately 2 billion people (26%) lacked safe drinking water, 3.6 billion people (46%) lacked safely managed sanitation and 2.3 billion people (29%) did not have access to a handwashing facility with soap and water (WHO, 2022, accessed on 29/8/22). Wastewater plants potentially play an important role in transmission of waterborne diseases due to re-introduction into water bodies or land application. Therefore, monitoring of water released from wastewater plants is crucial to manage the spread of waterborne pathogens. Several pathogens can be present in contaminated water, particularly waters containing faecal material; however, routine monitoring of all pathogens is not currently possible. Enterococcus faecalis, a microflora of human and animals has been used as a faecal indicator. Accurate and fast detection methods are critical for effective monitoring of E. faecalis in the environment. Although conventional and current molecular detection techniques provide sufficient sensitivity, specificity and throughput, their use is hampered by the 24-hour waiting period to obtain results, the need for expensive laboratory equipment, skilled personnel, and cold-chain storage devices. Therefore, we aimed to develop a simple, rapid and cost-effective detection platform which can be used at the point-of-need or resource-limited settings. An isothermal DNA amplification technique called recombinase polymerase amplification (RPA) that amplifies DNA at one constant temperature was integrated with a lateral flow assay (LFA) to detect E. faecalis with 100% specificity under 30 min without using any thermal cycling. The assay can detect 2.8×103 cells/100 mL E. faecalis from water and wastewater; 2.8×104 cells/100 mL from saline water. The developed assay can potentially be used for water monitoring from wastewater plants, during flood situations or in a resource-limited help in achieving some of the Sustainability Development goals.