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Background: Blood donation is crucial aspect of healthcare, ensuring steady supply of safe blood for medical treatments and emergencies. Understanding the beliefs, behaviors and opinions surrounding blood donation is essential for developing effective strategies to promote voluntary blood donation. This cross-sectional study aims to assess and analyze the factors influencing blood donation in Tumkur district of Karnataka, India. Methodology: A cross-sectional community-based study was conducted in Tumkur district over a period of six months. A total of 1000 subjects were included of age 18–65 years from different communities and socioeconomic backgrounds. The structured questionnaire was asked on demographics, knowledge, misunderstandings, previous donation history, willingness and decision-making variables. Results: The study found that while a majority (70%) of the participants were aware of the importance of blood donation, only small proportion (15%) had donated blood previously. Misconceptions and fear of needles were identified as key barriers to blood donation. Moreover, religious and cultural beliefs were observed to influence willingness to donate among certain communities. Younger age, higher education and positive experiences with blood donation drives were associated with an increased likelihood of future donation. Conclusion: Religious and cultural sensitivities should be taken into account when designing donation campaigns in specific communities. Engaging educational institutions and community organizations could play a pivotal role in promoting positive attitude towards blood donation among the youth. It is imperative for healthcare authorities to collaborate with local leaders and stakeholders to develop effective strategies for increasing voluntary blood donation rates in Tumkur district.