Many anthropogenic chemicals are manufactured and eventually enter the surrounding environment, threatening food security and human health. Considering the additive or synergistic effects of pollutant mixtures, there is an expanding need for rapid, cost-effective and field-portable screening methods in environmental monitoring. This study used a recently developed biospectroscopy-bioreporter-coupling (BBC) approach to investigate the binary toxicity of Ag(I), Cr(VI) and four organophosphorus pesticides (dichlorvos, parathion, omethoate and monocrotophos). Ag(I) and Cr(VI) altered the toxicity mechanisms of pesticides, explained by the synergistic or antagonistic effect of Ag/Cr-induced cytotoxicity and pesticide-induced genotoxicity. The discriminating Raman spectral peaks associated with organophosphorus pesticides were 1585 and 1682 cm−1, but 750, 1004, 1306 and 1131 cm−1 were found in heavy metal and pesticide mixtures. More spectral alterations were related to pesticides rather than Ag(I) or Cr(VI), hinting at the dominant toxicity mechanisms of pesticides in mixtures. Ag(I) supplement significantly increased the levels of reactive oxygen species induced by organophosphorus pesticides, attributing to the increased permeability of cell membrane and entrance of toxic substances into the cells by the oligodynamic actions. This study lends deeper insights into the interactions between microbes and pollutant mixtures, offering clues to assess the cocktail effects of multiple pollutants comprehensively.