Background Preprints are scientific manuscripts that are made available on open-access servers but are not yet peer-reviewed. Although preprints are becoming more prevalent, uptake is not optimal. Understanding researchers’ opinions and attitudes toward preprints is valuable to optimize their use. Understanding knowledge gaps and researchers’ attitudes toward preprinting can assist stakeholders, such as journals, funding agencies, and universities, to use preprints more effectively. Here, we aimed to collect perceptions and behaviours regarding preprints across an international sample of biomedical researchers. Methods Corresponding authors of articles published in biomedical research journals were identified from a random sample of journals from the MEDLINE database. Their names and email addresses were extracted to invite them to our anonymous, cross-sectional survey, which asked participants questions about their knowledge, attitudes, and opinions regarding preprinting. Results The survey was completed by 730 respondents providing a response rate of 3.20% and demonstrated a wide range of attitudes and opinions about preprints with authors from various disciplines and career stages worldwide. Most respondents were familiar with the concept of preprints but most had not previously posted one. The lead author of the project and journal policy had the greatest impact on decisions to post a preprint, whereas employers/research institutes had the least impact. Supporting open science practices was the highest ranked incentive, while increasing authors’ visibility was the highest ranked motivation for publishing preprints. Conclusions Although many biomedical researchers recognize the benefits of preprints, there is still hesitation among others to engage in this practice. This may be due to the general lack of peer review of preprints and little enthusiasm from external organizations such as journals, funding agencies, and universities. Future work is needed to determine optimal ways to improve researchers’ attitudes through modifications to current preprint systems and policies.