Agricultural practices, if not managed correctly, can have a negative impact on receiving environments via waste disposal and discharge. In this study, a chicken slaughter facility on the rural outskirts of Sydney, Australia, has been identified as a possible source of persistent effluent discharge into a peri-urban catchment. Questions surrounding the facility's environmental management practices go back more than four decades. Despite there having never been a definitive determination of the facility's impact on local stream water quality, the New South Wales Environment Protection Authority (NSW EPA) has implemented numerous pollution reduction requirements to manage noise and water pollution at the slaughter facility. However, assessment of compliance remains complicated by potential additional sources of pollution in the catchment. To unravel this long-standing conundrum related to water pollution we apply a forensic, multiple lines of evidence approach to delineate the origin of the likely pollution source(s). Water samples collected between 2014 and 2016 from irrigation pipes and a watercourse exiting the slaughter facility had elevated concentrations of ammonia (max: 63,000 μg/L), nitrogen (max: 67,000 μg/L) and phosphorus (max: 39,000 μg/L), which were significantly higher than samples from adjacent streams that did not receive direct runoff from the facility. Arsenic, sometimes utilised in growth promoting compounds, was detected in water discharging from the facility up to ˜4 times (max 3.84 μg/L) local background values (<0.5 μg/L), with inorganic As(∑V+III) being the dominant species. The spatial association of elevated water pollution to the facility could not unequivocally distinguish a source and consequently DNA analysis of a suspected pollution discharge event was undertaken. Analysis of catchment runoff from several local streams showed that only water sampled at the downstream boundary of the facility tested positive for chicken DNA, with traces of duck DNA being absent, which was a potential confounder given that wild ducks are present in the area. Further, PCR analysis showed that only the discharge water emanating from the slaughter facility tested positive for a generalized marker of anthropogenic pollution, the clinical class 1 integron-integrase gene. The environmental data collected over a three-year period demonstrates that the slaughter facility is indisputably the primary source of water-borne pollution in the catchment. Moreover, application of DNA and PCR for confirming pollution sources demonstrates its potential for application by regulators in fingerprinting pollution sources.