There is considerable current interest in using biochar (BC) as a soil amendment to sequester carbon to mitigate climate change. However, the implications of adding BC to agricultural soil for the environmental fate of pesticides remain unclear. In particular, the effect of biochars on desorption behavior of compounds is poorly understood. This study examined the influence of BC on pesticide chemical and biological accessibility using the herbicide isoproturon (IPU). Soils amended with 1% and 2% BC showed enhanced sorption, slower desorption, and reduced biodegradation of IPU. Addition of 0.1% BC had no effect on sorption, desorption or biodegradation of IPU. However, the mineralization of 14C-IPU was reduced by all BC concentrations, reducing by 13.6%, 40.1% and 49.8% at BC concentrations of 0.1%, 1% and 2% respectively. Further, the ratio of the toxic metabolite 4-isopropyl-aniline to intact IPU was substantially reduced by higher BC concentrations. Hydroxypropyl-β-cyclodextrin (HPCD) extractions were used to estimate the IPU bioaccessibility in the BC-amended soil. Significant correlations were found between HPCD-extracted 14C-IPU and the IPU desorbed (%) (r2 = 0.8518, p < 0.01), and also the 14C-IPU mineralized (%) (r2 = 0.733; p < 0.01) for all BC-amended soils. This study clearly demonstrates how desorption in the presence of BC is intimately related to pesticide biodegradation by the indigenous soil microbiota. BC application to agricultural soils can affect the persistence of pesticides as well as the fate of their degradation products. This has important implications for the effectiveness of pesticides as well as the sequestration of contaminants in soils.