Water is the most important ingredient of life. Water fecal pollution threatens water quality worldwide and has direct detrimental effects on human health and the global economy. Nowadays, assessment of water fecal pollution relies on time-consuming techniques that often require well-trained personnel and highly-equipped laboratories. Therefore, faster, cheaper, and easily-used systems are needed to in situ monitor water fecal pollution. Herein, we have developed colorimetric lateral flow strips (LFS) able to detect and quantify Escherichia coli species in tap, river, and sewage water samples as an indicator of fecal pollution. The combination of LFS with a simple water filtration unit and a commercially available colorimetric reader enhanced the assay sensitivity and enabled more accurate quantification of bacteria concentration down to 10⁴ CFU mL⁻¹ in 10 minutes, yielding recovery percentages between 80% and 90% for all water samples analyzed. Overall, this system allows for monitoring and assessing water quality based on E. coli species as a standard microbiological indicator of fecal pollution. Furthermore, we have developed a novel bioluminescent, bacteria-based method to quickly characterize the performance of a great variety of LFS materials. This new method allows evaluating the flow rate of big analytes such as bacteria through the LFS materials, as a suggestive means for selecting the appropriate materials for fabricating LFS targeting big analytes (≈2 μm). As a whole, the proposed approach can accelerate and reduce the costs of water quality monitoring and pave the way for further improvement of fecal pollution detection systems.