With respect to their adverse health and environmental effects, halogenated organic compounds (HOCs) are among the most important priority pollutants. HOCs cannot be easily biodegraded. However. if suitable microbial cultures are acclimated to these compounds properly, and the optimum operating conditions are achieved, HOCs can be removed with biological methods. Recently, great interest has focused on reductive anaerobic dehalogenation for the removal of these compounds. This process yields lower halogenated compounds which are less toxic and more amenable to further biodegradation. Chloroacetic (CAA), trichloroacetic (TCAA), and 2,4-dichlorophenoxyacetic acids (2,4-D) are significant industrial compounds mainly used in herbicide, pesticide, and dye manufacturing. The objective of this study was to: (i) investigate the inhibitory effects of CAA, TCAA, and 2,4-D; and (ii) determine the optimum loading strategy of these HOCs to anaerobic cultures. To this purpose Anaerobic Toxicity Assay (ATA) and Optimum Loading Strategy experiments were conducted for CAA, TCAA, and 2,4-D, The results of this study are expected to develop an optimum loading strategy of HOCs to anaerobic systems and contribute to more effective use of anaerobic reductive dehalogenation of HOCs.