The influence of biochar (5%) on the loss, partitioning and bioaccessibility of 14C-isoproturon (14C-IPU) was evaluated. Results indicated that biochar had a dramatic effect upon 14C-IPU partitioning: 14C-IPU extractability (0.01 M CaCl2) in biochar-amended treatments was reduced to <2% while, 14C-IPU extractability in biochar free treatments decreased with ageing from 90% to 40%. A partitioning model was constructed to derive an effective partition coefficient for biochar:water (KBW of 7.82 × 104 L kg−1). This was two orders of magnitude greater than the apparent Kfoc value of the soil organic carbon:water (631 L kg−1). 14C-radiorespirometry assays indicated high competence of microorganisms to mineralise 14C-IPU in the absence of biochar (40.3 ± 0.9%). Where biochar was present 14C-IPU mineralisation never exceeded 2%. These results indicate reduced herbicide bioaccessibility. Increasing IPU application to ×10 its recommended dose was ineffective at redressing IPU sequestration and its low bioaccessibility. Display omitted •Biochar had a dramatic effect on IPU partitioning.•IPU extractability was reduced to <2% in biochar treatments.•Effective partition coefficient for biochar:water (KBW) was 7.82 × 104 L kg−1.•KBW was 124 times greater than the apparent Kfoc value of the control.•Biochar precluded microbial bioaccessibility – no catabolic response was observed. Biochar dramatically reduced 14C-IPU extractability (<2%) with KBW being ×123 greater than the apparent Kfoc. Correspondingly, microbial bioaccessibility of IPU was negligible.