The Sawyer PointONE™ hollow fiber membrane filter is increasingly promoted for long-term household water treatment in developing countries. Limited data demonstrate PointONE™ microbiological laboratory efficacy and short-term diarrheal disease reduction among users, but household microbiological data is lacking. To compare laboratory and household PointONE™ filter microbiological performance, we enumerated Escherichia coli (E. coli) and total coliforms in source and filtrate water from: (1) one new filter with E. coli-spiked water (107–109 CFU/100 mL) in the laboratory, (2) one new filter with natural Maine and Honduran surface waters, and (3) 50 filters used in Honduran homes for 1–3 years. In laboratory tests, all filtrate samples had <1 CFU/100 mL E. coli (>99.99999% reduction). In natural surface waters, all filtrate samples had ≤1 MPN/100 mL E. coli (≥99.5% reduction). In households, filtrate samples had geometric mean 5.1 MPN/100 mL E. coli (90% reduction), with only 30% of filtrate samples complying with international standards of undetectable E. coli. Total coliform presence in natural water filtrate varied for both new and household filters. The discrepancy between laboratory and household results and premature filter failure are not well understood. Further research is recommended to understand this performance disparity and determine filter failure mechanisms in households.