Fecal microbiota transplantation (FMT) is effective against recurrent Clostridioides difficile infection (rCDI), but its safety is jeopardized by the potential transmission of pathogens, so international guidelines recommend either a quarantine or a direct stool testing. Whereas reports of the quarantine-based approach are emerging, data on the direct testing-based approach are not available. Our aim is to report outcomes of a donor screening framework for FMT including direct stool testing. In this prospective cohort study, all donor candidates recruited at our FMT centre underwent a four-step screening process to be enrolled as actual donors. Each collected stool donation was then evaluated with a direct stool testing including a molecular assay for gut pathogens and a culture assay for multi-drug resistant organisms (MDRO). From January 2019 to June 2023, 72 of 227 candidates (32%) were considered eligible and provided 277 stool donations. Ninety-nine donations (36%) were discarded for positivity to intestinal pathogens, most commonly enteropathogenic Escherichia coli (n = 37) and Blastocystis hominis (n = 20). Overall, 337 stool aliquots were obtained from 165 approved donations. All suspensions were used for patients with rCDI, and no serious adverse events or clinically evident infections were observed at 12 weeks after procedures. In our study, screening of donor faeces including direct stool testing led to the discard of a considerable rate of stool donations but was also extremely safe. This approach may represent a reliable strategy to guarantee the safety of FMT programs, especially in countries with high prevalence of MDRO.