Background: Transfusion-transmissible infections (TTIs) remain a significant concern forblood safety, particularly in resource-limited settings. Nucleic acid testing (NAT) can detect infections during the window period before seroconversion, offering advantages over conventional serology. This study compared the efficacy of serological and molecular methods for detecting TTIs among blood donors in Bangladesh. Methods: A cross-sectional study was conducted at the Department of Transfusion Medicine, Shaheed M. Monsur Ali Medical College, Sirajganj, from January to December 2025. A total of 150 blood donors were enrolled through consecutive sampling. Blood samples were tested using both serological (ELISA for HBsAg, Anti-HCV, Anti-HIV, and malaria antigen) and molecular (real-time PCR for HBV DNA, HCV RNA, and HIV RNA) methods. Window period infections were identified as serology-negative but NAT-positive cases. Data were analyzed using SPSS version 26. Results: The study population was predominantly male (92.0%) and young adults (mean age 31.5 years). Voluntary donors comprised 65.3% of the sample. Overall TTI prevalence was 15.3% (23/150), with HBV most common (8.0%), followed by HCV (4.0%) and HIV (2.7%). NAT detected 25% more infections than serology (20 vs. 16), with significant differences for HBV (p=0.023), HCV (p=0.045), and HIV (p=0.032). Seven window period infections (4.7% of donors) were detected by NAT but missed by serology, including 3 HBV, 2 HCV, and 2 HIV cases. Serology demonstrated poor sensitivity (65.0%) but excellent specificity (99.3%). McNemar’s test confirmed significant overall difference between methods (p=0.023), with substantial agreement (ê=0.78). Conclusion: NAT detects significantly more TTIs than serology alone among Bangladeshi blood donors, with 7 window period infections identified that would have been missed by routine screening. Serology misses 35% of active infections, leaving unacceptable residual risk. These findings support phased implementation of NAT to improve blood safety in Bangladesh.