Background: Bloodstream infections necessitate rapid identification and management. Automated blood culture systems have revolutionized diagnostic microbiology by expediting pathogen detection and enabling effective clinical interventions. This study assesses the optimal incubation period for blood cultures in a tertiary healthcare setting, aiming to enhance diagnostic accuracy and support antibiotic stewardship. Objective: To evaluate the adequacy of incubation times in blood cultures for producing clinically significant results using an automated system in a tertiary care setting. Methods: This cross-sectional descriptive study was conducted at the Microbiology Section of Combined Military Hospital, Lahore, from October 2022 to March 2023. All patient blood samples processed through the Bactec/Alert system were studied. Positive samples were cultured on Blood and MacConkey agar and incubated at 35 ± 2°C for 24 hours. Positivity rates, contamination, and patient demographics were analyzed using the Laboratory Information Management System. Results: Of the 8,216 blood culture bottles processed, 490 (5.96%) were flagged as positive. Early detection within the first 24 hours was noted in 41.63% (n=204) of cases, with subsequent detections on the second day at 58.65% (n=258), the third day at 5.30% (n=26), and the fourth day at 0.20% (n=1). The isolated pathogens included gram-positive organisms (16.32%, n=80), gram-negative organisms (80.20%, n=393), and Candida spp. (3.46%, n=17). Conclusion: Timely and effective blood culturing is crucial for the diagnostic confirmation of bacteremia, enabling appropriate pathogen identification and antimicrobial therapy. Shortening detection times can significantly impact clinical outcomes, reduce healthcare costs, and promote antimicrobial stewardship.