Background: Donating blood can help in saving the lives of many individuals around the globe. Evidence indicates that there is a shortage of blood and blood products in many countries. This study aimed at assessing knowledge, attitudes, and practices towards blood donation and its associated factors. Methodology: A community-based and cross-sectional study was conducted at Najran University from November 2020 to June 2021. A convenient sampling technique was adopted for recruiting 445 participants. A structured electronic questionnaire was utilized for collecting data. Chi-square and logistic regressions were carried out. Associations were considered significant when pvalue< 0.05. Results: In this study, 310 (62%) were males. 205 (46.1%) are currently studying in health colleges. 272 (61.1%) were found to be knowledgeable, while 265 (59.6%) were having favorable attitudes towards blood donation respectively. Only 98 (22%) of the participants reported having an experience of blood donation. Health colleges’ participants were significantly having good knowledge regarding blood donation. Studying field, gender, and previous experience of blood donation were found to be independent predictors of attitude. Conclusion: The level of knowledge regarding blood donation was found to be reasonable. The findings of the current study also justified any possible and regularly scheduled campaigns for raising awareness regarding voluntary donation.