This study investigated the potential application of allyl isothiocyanate (AITC) for the disinfection of water by examining the bactericidal effects of this compound against inoculated Escherichia coli in autoclaved, de-chlorinated tap water and against the natural microbiota (heterotrophic plate count bacteria, HPCs) in a surface water. Bacteriostatic trials for AITC demonstrated growth inhibition against E. coli in tryptone soy broth, with AITC concentrations of 47.5, 63.3 and 126.6 mg/l inhibiting growth over two hours of contact time and 505.5 mg/l achieving complete growth inhibition for seven days. These AITC concentrations were then used to assess the bactericidal effect of AITC in water matrices, through which at least a 1.22-log10 reduction of both E. coli and HPCs was achieved in all trials, and up to a maximum of 2.93-log10 reduction of E. coli with 126.6 mg/l of AITC and two hours of contact time. AITC was consistently less effective against the HPCs in the surface water compared to the inoculated E. coli in tap water. Under the conditions that were tested, AITC was unable to reduce the HPC values below 100 CFU/ml, the World Health Organization guideline for safe drinking water, suggesting that AITC should not be used as a potable water disinfectant on its own; however, there may be applications for AITC as a biocide for non-potable water storage or treatment.