As a cost-effective sorbent, modified biochar has received increasing attention for the removal of heavy metal contaminants. Among several chemical modification methods, introducing thiol functional groups onto the surface of biochar has been identified as an effective enhancement approach for the heavy metal sorption and removal capacity of this porous adsorbent material. In general, chemical impregnation is a widely used method to graft thiol groups onto the surface of carbon-based materials. However, limited comparative data are available on the efficacy of the present biochar thiolation methods. In this study, the biochar of nine different organic sources was modified by two frequently used agents with distinct thiolation mechanisms: 3-Mercaptopropyltrimethoxysilane (3-MPTS) and β-mercaptoethanol. In addition to chemical impregnation, the ball milling method, a simple and environmentally friendly alternative thiolation method, was also evaluated. A comprehensive structural characterization of the biochar samples was completed before and after thiolation. A higher concentration of sulfur on the surface of the biochar was achieved through thiolation with β-mercaptoethanol, in which the thiolation mechanism is performed through an esterification reaction with the carboxylic acid functional groups of the activated biochar. Chemical impregnation was found to be a more effective thiolating method than ball milling using the same thiolating agent.