Biochar application can improve soil quality, but at high rates may cause negative effects on soil organisms such as earthworms. Conversely, as ecosystem engineers and catalysts of microbial activity, earthworms can also affect soil quality and interact with biochar, though little is known concerning endogeic earthworm species interactions with biochar. Hence, a standard ecotoxicological laboratory trial was undertaken to assess the impact of Brazil-nut husk biochar application (0, 2.5, 5 and 10% w/w) on the endogeic cosmopolitan species Pontoscolex corethrurus, and the effects of bioturbation and gut passage on biochar chemical characteristics (assessed using pyrolysis coupled with gas chromatography and mass spectrometry) and morphology (evaluated using scanning electron microscopy). Biochar addition up to 10% w/w had no negative effects on P. corethrurus, and ingested biochar particles showed signs of physical degradation and bacterial colonization. Nine pyrolysis-produced chemical biomarkers (phenolic and aromatic compounds) were detected that can be used as indicators of biochar presence in soils and earthworm castings. Earthworm casts showed distinct macromolecular chemical signatures compared with uningested artificial soil with and without biochar. Earthworm bioturbation and biochar ingestion may have important effects on functionalization and biochar chemical and physical properties that should be considered in field applications, especially where endogeic biochar-consuming species are present.