Marburg virus (MARV) nucleic acid was detected in Rousettus aegyptiacus bats in 2005 and 2006 in the midwest and southeast of Gabon. In this study we used MARV-specific real-time reverse-transcription polymerase chain reaction (RT-PCR) and MARV-specific nested RT-PCR assay to screen 1257 bats caught during July 2009, December 2009, and June 2010 in 3 caves situated in northern Gabon. Nine specimens tested positive by the real-time assay, with cycle threshold values ranging from 35 to 39, of which only 1 R. aegyptiacus specimen collected in 2009 was positive in the nested VP35 RT-PCR assay. Together with MARV-positive bats in the south and west found in 2005 and 2006, confirmation of phylogenetically closely related MARV-positive bats 5 years later and in northern Gabon suggests that MARV is now enzootic in Gabon and emphasizes the importance of long-term monitoring of bat populations and human-bat interfaces.