In microbiological water quality testing, sample dechlorination with sodium thiosulfate is recommended to ensure that results accurately reflect the water quality at sample collection. Nevertheless, monitoring institutions in low-resource settings do not always dechlorinate samples, and there is limited research describing how this practice impacts drinking water quality results. The effect of dechlorination on indicator bacteria counts was evaluated by spiking laboratory water with five Escherichia coli (E. coli) concentrations (104–108 CFU/100 mL), chlorinating at six doses (0–0.6 mg/L), holding samples with and without sodium thiosulfate for 5–7 hours, and enumerating E. coli by membrane filtration with m-lauryl sulfate media. Additionally, sub-Saharan African water suppliers enumerated thermotolerant coliform by membrane filtration in paired chlorinated water samples collected with and without sodium thiosulfate. Across all E. coli and chlorine doses in the laboratory, and all field tests, samples held without sodium thiosulfate had lower bacteria counts (p < 0.001). Additionally, chlorinated water supply samples held without sodium thiosulfate had an 87.5% false negative rate. Results indicate the importance of dechlorinating microbiological water quality samples, discarding data from chlorinated samples collected without dechlorination, and reinforcing dechlorination recommendations in resource-limited environments to improve water safety management.