Nanoliquid chromatography (nanoLC) was coupled to high-resolution mass spectrometry (HRMS) to perform a non-targeted investigation on benthic invertebrates, Chironomus riparius exposed to wastewater treatment plant (WWTP) effluents. Insect larvae represent a complex and low-weight matrix that required the use of a miniaturized Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method of extraction followed by nanoLC-HRMS to perform the analysis. The optimization of this coupling in terms of separation conditions including trapping step, detection conditions and data treatment provided reproducible fingerprints on insect larvae exposed to WWTP effluents with both in situ and ex-situ approaches. Statistical treatments such as principal component analysis highlighted the impact of WWTP effluents on the metabolome of insect larvae and showed the influence of exposure conditions. The identification of discriminating signals (m/z, tR) matched with several potential endogenous biomarkers. These are mainly fatty acids, indicating a change in lipid metabolism that can be correlated with exposure to WWTP effluents. Several xenobiotics have also been detected, including ibuprofen and propranolol, whose identities have been confirmed by analytical standards. This work demonstrates the effectiveness and sensitivity of nanoLC-HRMS based environmental non-targeted approaches in ecotoxicological studies and provides the first profiling data for a very small aquatic invertebrate.