Bloodstream infections are associated with high morbidity and mortality. Blood culture remains the gold standard for diagnosis. Inadequate incubation of the solid media subculture plates may result in delayed diagnosis. This study aimed to evaluate the diagnostic yield of extended incubation of subculture plates from conventional liquid blood cultures, analyse the recovered isolates, and determine the rate of contamination during extended incubation. This prospective, laboratory-based observational study was conducted in the Department of Microbiology at a tertiary care teaching hospital from January to March 2025. A total of 851 one-day-old sterile subculture plates were further incubated at 37 °C for another five days (i.e., day 2 to day 6) and observed daily for growth of potential pathogenic bacteria or visible contamination. Plates showing visible contamination were discarded, while those without contamination were further processed for identification of potential bacterial pathogens using routine laboratory procedures.  Extended incubation yielded 29 additional potential pathogens (3.4%), predominantly Enterococcus spp. (41.5%), Acinetobacter spp. (20.7%), Staphylococci other than Staphylococcus aureus (20.7%) and Candida tropicalis (6.9%). Plate contamination rates after six days were 13.4% for blood agar and 9.0% for MacConkey agar. Contamination remained within acceptable limit (≤5%) after three days of incubation for both agar and after four days for only MacConkey agar, indicating a safe diagnostic window for extended incubation. Prolonging incubation can improve the detection of slow-growing pathogens with minimal contamination risk, even after the third or fourth day of incubation. This low-cost strategy is particularly relevant for resource-limited settings and could prevent missed diagnoses.