Background: Blood donation is essential for healthcare systems worldwide, yet maintaining an adequate donor pool remains challenging, particularly in developing countries like Bangladesh where only 30% of blood comes from voluntary donors. Understanding psychosocial and sociocultural factors influencing donor behavior is crucial for developing effective recruitment strategies. Objective: This study aimed to assess motivational factors, barriers, and sociocultural influences affecting blooddonation behavior among donors and non-donors in Sirajganj, Bangladesh. Methods: A cross-sectional comparative study was conducted from January to June 2024 at the Department of Transfusion Medicine, Shaheed M. Monsur Ali Medical College, Sirajganj. A total of 200 participants (100 donors, 100 non-donors) were enrolled using purposive sampling. Data were collected through face-to-face interviews using a structured questionnaire covering sociodemographics, motivations, barriers, and sociocultural beliefs. Statistical analysis was performed using SPSS version 26 with descriptive statistics, chi-square tests, t-tests, and binary logistic regression. Results: Donors were predominantly male (91.0% vs. 66.0%, p<0.001), educated (64.0%vs. 44.0%, p=0.012), and urban residents (56.0% vs. 42.0%, p=0.020). Altruistic motivation was the strongest domain (mean 4.40), with 92.0% reporting satisfaction from helping others. Among non-donors, primary barriers were lack of awareness (48.0%), lack of time (46.0%), fear of fainting (44.0%), and misconceptions about weakness (37.0%). Donors demonstrated significantly higher knowledge (3.42 vs. 2.18, p<0.001), greater public trust (4.12 vs. 3.24, p<0.001), and stronger social support (3.86 vs. 3.12, p<0.001). Independent predictors of donation were male gender (AOR=6.17), knowledge of blood group (AOR=5.37), public trust (AOR=3.45), knowledge score (AOR=2.89), and higher education (AOR=2.14). Conclusion: Blood donation in Sirajganj is driven primarily by altruism, intrinsic rewards, and religious beliefs, while lack of awareness, fear, and mistrust are major barriers. Public trust emerged as a critical independent predictor. Targeted interventions addressing knowledge gaps, leveraging religious motivation, and building institutional trust are essential for increasing voluntary blood donation in Bangladesh.