Biochar (BC) has been widely used for soil remediation and pollutant removal from environmental water. The pollutant-adsorbing BC often exerts different cytotoxicity from pollutant-free BC. However, how adsorption of multiple pollutants alters the cytotoxicity of BC and how to modulate such toxicity are still unclear. By preparing BCs from two different materials (Banana peel (BP) and corn stalk (CS)) at two pyrolysis temperatures (300 °C and 500 °C, namely BP300-BC, BP500-BC, CS300-BC, and CS500-BC, respectively), we systemically investigated factors that affect the combined cytotoxicity of pollutant-adsorbing BC in Human normal rectal mucosal cells (FHC). The reduction of Cr(VI) to Cr(III) occurred on BC, in particular on BC prepared at 300 °C, in which larger amount of organic matters were left. The presence of Cu(II) promoted the release of Cr from BC once entering cells, which was more obvious for BC prepared at 500 °C. The changes in valence states and release rates of Cr adsorbed on BC prepared at different pyrolysis temperatures together caused reduced cytotoxicity of BP300-BC-Cr-Cu but enhanced cytotoxicity of BP500-BC-Cr-Cu, compared to the same amount of mixture of Cr(VI)–Cu(II). Our findings offer insight understanding of how pyrolysis temperature and the adsorbed multiple pollutants affect the combined cytotoxicity of BC-pollutant complex, allowing for safe applications of BC in future.