Background The change from non-molecular to nucleic acid amplification tests (NAATs) is known to increase the detection of Clostridium difficile infection (CDI); however, the impact on stool rejection policies in clinical laboratories is unclear. The current guidelines have reinforced the importance of respecting strict conditions for performing tests on stool samples for CDI diagnosis. The purpose of this study was to estimate whether the implementation of molecular tests has resulted in changes in stool rejection policies between clinical laboratories that introduced NAATs and those that did not. Results A survey was conducted to evaluate the change in the number of stool samples rejected and the rejection criteria among 12 hospital laboratories in southwestern France before and after the switch from non-molecular tests to NAATs using retrospective data from June 1 till September 30, 2013 and the same period 2014. Four laboratories introduced NAATs as a second or third step in the process. A total of 1378 and 1297 stools samples were collected in 2013 and 2014, respectively. The mean number of rejected stool samples significantly increased (p < 0.001, Chi square test), with a total of 99 (7.1%) and 147 (11.3%) specimens rejected in 2013 and 2014, respectively. Notably, these laboratories had more stringent criteria and were no longer testing the stool samples of patients with CDI-positive results within 7 days. In contrast, there was a significant decrease in the rate of rejected stool samples (p < 0.001, Chi square test) in the five laboratories that did not adopt NAATs and a less stringent stool rejection policy. Conclusion Nucleic acid amplification test implementation improved compliance with recommended stool rejection policies. Laboratories should follow the recommended laboratory algorithm for the CDI diagnosis combined with the correct stool rejection policy.