We evaluated retrospectively the yield of stool culture (SC) depending on the length of hospitalization, and we characterized the patients missed by the 3-day rejection rule. SC detects bacterial enteric pathogens (Campylobacter spp., Salmonella enterica, Yersinia spp., Shigella spp.). During this 5-year study period, 13,039 SCs were requested, and 376 were positive (2.9%). The yield of SC dropped from 11.7% before 3 days of hospitalization to 0.7% after 3 days in children and 4.3% to 0.3% in adults. Finally, only 13 clinically relevant cases (0.2% of SC prescribed after 3 days) were undiagnosed by strict application of the 3-day rule. In conclusion, rejection of SC prescribed after 3 days of hospitalization allows to reduce workload by 37.8% for children and 65.7% for adults, representing a cost of euro12,500 ($16,250) per year in our hospital.