Background and Aims: Blood donation (BD) is described as altruistic behavior and one of the life-saving practices. The deficit of blood supply from BD has become a global concern. Knowledge, attitude, practice, and motivation play a key role in BD. This study is aimed to determine the factors that hinder the healthy individuals who have not donated blood yet from BD, by exploring the demographic and other variables among the citizens of Jeddah city, Kingdom of Saudi Arabia (KSA). Methods: A cross-sectional descriptive study was carried out at Faculty of Medicine, King Abdul-Aziz University (KAU), Jeddah, KSA, between June 01, 2016 to June 30, 2016. The study included 327 participants who were selected by convenient random sampling in the Jeddah City. The data was collected by using the online distribution of the previously validated questionnaire. Results: Half of the participants (51.1%) have good knowledge about BD. More than two-thirds of participants showed a positive attitude towards BD. Majority of participants (87.7%)   claimed that they will donate blood if the recipient is a friend or relative. The most chosen reasons for not donating blood among participants were BD did not cross their minds (23.9%), no time for BD (17.1%) and fear of needles (13.8%). Mobile blood campaign (84.4%) and one day off (83.2%) were important motivating factors for most of the participants. Almost half of the participants (52.29%) mentioned that BD should be non-remunerable, 26.61% agreed that remuneration is a motive and 21.10% believe that any form of gift is acceptable as a motivating factor for BD. Conclusion: Although most of the participants have good knowledge and positive attitude towards BD, still numerous potential and eligible donors evade BD. More awareness and motivational programmes are required to bring more individuals to the pool of regular donors.