Purpose To assess the additional value of blood culture bottles (BCBs) in the diagnosis of endophthalmitis by comparing its culture yield with that of conventional media (CM). Design Retrospective consecutive case series. Methods We included patients who were treated between January 2001 and January 2010 for clinically suspected endophthalmitis of any etiology, and had vitreous specimens cultivated in both BCB and CM. Results Specimens from 85 eyes from 85 patients were included. The culture yield of BCB was 69%, and that of CM was 72% (difference not significant). Adding the results of BCB improved the yield of CM significantly by 13%, resulting in a combined yield of 81%. The sensitivity of detection of Haemophilus influenzae in BCB seemed lower compared with CM, possibly due to the lack of growth factors in the BCB. There was no difference in yield between specimens obtained by tap or by vitrectomy. Conclusion In contrast with earlier reports, we did not find BCB superior to CM. The combined use of BCB and CM increased the pathogen detection rate significantly and should therefore be considered as the microbiological method of choice in the work-up of endophthalmitis.