Drinking water biofilters can improve water quality by transforming contaminants or their precursors, but they also can develop headloss more rapidly than do abiotic filters. Phosphorus supplementation has been proposed as one strategy to lengthen biofilter run times, but the impact of this strategy in field tests has been mixed. The current bench-scale study found that severe phosphorus limitation, as indicated by a high phosphatase to total glycosidase activity ratio (PHO:GLY), led to 230% higher headloss accumulation rate when particles were loaded onto the biofilters as compared to the same experiment performed under a mild phosphorus limitation. Phosphorus limitation was associated with higher concentrations of extracellular polymeric substances, lower biomass concentrations, a more filamentous biofilm morphology, and increased relative abundance of Hyphomicrobiaceae (a family of stalked bacteria) on the biofilter media. These differences in the biofilm likely contributed to higher headloss. This work suggests that phosphorus supplementation could improve biofilter hydraulics in the field if the biofilter is severely phosphorus limited, which was indicated by a PHO:GLY greater than 154 under the conditions tested in this study.