BACKGROUND: The pre-storage condition of blood culture bottles prior to entering the automated blood culture system may affect the time to detection (TTD) of microorganisms and the final report days. METHODS: We compared the TTD and final report days according to the pre-incubation conditions after laboratory operating hours: room temperature (RT) vs. a BacT/Alert unit (BioMerieux Inc.) for 3 months respectively. All bottles were inserted into the main BacT/Alert system the next morning. RESULTS: TTD was significantly reduced by pre-incubating bottles in a BacT/Alert unit (median, 8.4 h) compared to pre-storage at RT (median, 12.4 h) (P< 0.001). The final report of bacterial identification and drug susceptibility within 2 days was available for 24.4% of bottles pre-incubated in a BacT/Alert unit compared to 14.9% of those incubated at RT. The false positive results were significantly higher for pre-incubation in a BacT/Alert unit (0.81%) than for that (0.29%) at RT (P<0.001). CONCLUSION: If a clinical microbiology laboratory is not operational for 24 hours, an automated blood culture unit might be a good alternative to reduce TTD and allow the submission of a faster final report compared to pre-storage at RT. However, false positive readings increased more than two-fold by pre-incubation in a BacT/Alert unit.