The utilization of biowastes for producing biochar to remove potentially toxic elements from water represents an important pathway for aquatic ecosystem decontamination. Here we explored the significance of thiol-functionalization on sugarcane bagasse biochar (Th/SCB–BC) and rice husk biochar (Th/RH–BC) to enhance arsenite (As(III)) removal capacity from water and compared their efficiency with both pristine biochars (SCB–BC and RH–BC). The maximum As(III) sorption was found on Th/SCB–BC and Th/RH–BC (2.88 and 2.51 mg g−1, respectively) compared to the SCB–BC and RH–BC (1.51 and 1.40 mg g−1). Relatively, a greater percentage of As(III) removal was obtained with Th/SCB–BC and Th/RH–BC (92% and 83%, respectively) at a pH 7 compared to pristine SCB–BC and RH–BC (65% and 55%) at 6 mg L−1 initial As(III) concentration, 2 h contact time and 1 g L−1 sorbent dose. Langmuir (R2 = 0.99) isotherm and pseudo-second-order kinetic (R2 = 0.99) models provided the best fits to As(III) sorption data. Desorption experiments indicated that the regeneration ability of biochars decreased and it was in the order of Th/SCB–BC (88%) > Th/RH–BC (82%) > SCB–BC (77%) > RH–BC (69%) up to three sorption–desorption cycles. Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy results demonstrated that the thiol (-S–H) functional groups were successfully grafted on the surface of two biochars and as such contributed to enhance As(III) removal from water. Spectroscopic data indicated that the surface functional moieties, such as -S–H, − OH, − COOH, and C = O were involved to increase As(III) sorption on thiol-functionalized biochars. This study highlights that thiol-grafting on both biochars, notably on SCB–BC, enhanced their ability to remove As(III) from water, which can be used as an effective technique for the treatment of As from drinking water.