Agricultural water is a known vector for the transfer of foodborne pathogens onto fresh produce. Development of pre-harvest and post-harvest microbial profiles of agricultural water used by fresh produce growers, processors, and holdings is a requirement under the Food Safety Modernization Act Produce Safety Rule. One of the United States Environmental Protection Agency (US EPA) approved agricultural water testing methods is US EPA Method 1603, which requires no greater than a 6-h time frame between the collection of the water sample and initiation of analysis. This 6-h timeframe is unrealistic for many produce growers due to there being few laboratories certified to conduct testing and the geographic location of the farms. Agricultural water samples (n = 101) from well water and surface water were collected from 60 different farms to determine if holding samples for 24 h yielded significantly more generic Escherichia coli (E.coli) than 6 h using EPA 1603 method. A total of 32 samples were found contaminated with generic E. coli. Of these positive samples, surface water accounted for 87.5% of the samples (n = 28). There was no significant disparity between populations of generic E. coli at 6- and 24-h sample-test time interval (p > 0.05). These results provide evidence that the sample-test time interval can be extended to 24-h time, which makes quantitative generic E. coli testing for agricultural water as mandated by the FSMA Produce Safety Rule more accessible to growers.