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Soil-dwelling insects may be at particularly high risk of exposure to pesticides because soils are a major recipient of these chemicals. Still, we lack non-invasive techniques to detect in situ responses of soil macrofauna to pesticides and other contaminants. Here, we test if sound recordings can reveal sub-lethal behavioural shifts in ant colonies caused by a neonicotinoid (imidacloprid). We demonstrate that an acoustic variable can detect pesticide-induced shifts in outdoor behaviours (increased activity) of red wood ants (Formica rufa). This behaviour shift was observed at environmentally relevant, sub-lethal levels of imidacloprid (10.8 ± 5.7 ng g-1 sample, average ± standard error). Notably, our recordings revealed a short-term (<6 h) increase in activity induced by the pesticide, followed by a slow recovery and a return to the baseline over a weekly timescale. The long-term sub-lethal behavioural effects would have been difficult to detect using other techniques, given the opaque nature of the studied mounds. We conclude that acoustic monitoring offers a promising tool for detecting pesticide effects in natural settings. We also provide recommendations for best practices for acoustic monitoring of soil fauna and discuss the challenges of measuring soil fauna behavioural changes in a natural setting.