Blood collection agencies (BCAs) hosting stool (fecal or poo) donor programs report high rates of donor deferral. However, the impact of deferral on willing donors, in terms of personal well-being and future engagement with BCAs, remains unexplored. Accordingly, we surveyed those attempting to donate intestinal microbiota about their experience of being ineligible. A total of 196 potential stool donors from Australia's BCA (>90% blood/blood product donors) completed the first stage of eligibility screening and then an online survey once notified of their ineligibility. Respondents reported motives for donating, perceptions of screening and improvements needed, experience of being told they are ineligible, and their feelings about this. Over 80% of participants were ineligible to donate. Of those ineligible, 58% did not know why they were ineligible resulting in potentially future eligible donors being permanently lost. Motives (>5%) included helping others, being a human substance donor, understanding benefits, curiosity/novelty, and helping science/research. Participants identified they needed clear and timely information during screening and a specific reason for their ineligibility. Participants commonly experienced disappointment, confusion, and calm in response to being ineligible. BCAs need strategies to mitigate the disappointment of ineligible donors, maintain satisfaction with BCAs, and preserve donor identity since many ineligible donors give multiple human substances. BCAs should provide more information about eligibility criteria during early screening stages to reduce disappointment and give personalized information about ineligibility to resolve the confusion. Offering alternative opportunities to give may reduce disappointment and increase ineligible donor engagement.