Nowadays, many commercial kits allow the detection of sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts' DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts' DNA extraction. A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the oocyst' DNA extraction, before amplification using the same real-time PCR method targeting. The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of oocysts (i.e., 0-94.4% and 33.3-100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24 with Lysing Matrix E . Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the DNA amplification methods.