Two bench-scale biofiltration columns were monitored to examine the influence of water-quality parameters, including pH and alkalinity to enhance drinking water biofiltration efficiency. The biofilters were operated at four pH values with low and high alkalinity levels. Applying a higher pH level of 7.5 compared to 6.0 led to similar total organic carbon (TOC) removal efficiency. Raising the pH to 10.0 resulted in a significantly lower TOC removal efficiency. Increasing pH was also observed to influence ammonia removal significantly such that ammonia removal efficiency improved from 13% at pH 6.0 to 93% at pH 10.0; however, the higher pH was no longer attributed to biological removal but ammonia stripping. The assessment of theoretical oxygen demand revealed that dissolved oxygen (DO) availability was an influential factor in nitrification efficiency. No direct correlation was observed between adenosine triphosphate (ATP) and TOC removal. Overall, pH 7.5 demonstrated optimal biofilter conditions.