Benthic nitrogen fixation has been estimated to contribute 15 Tg N year1to themarine nitrogen budget. With benthic marine nitrogen fixation being largelyoverlooked in more recent surveys, a refocus on benthic diazotrophy wasconsidered important. Variations in nitrogenase activity (acetylene reduction-gaschromatography) in a tropical lagoon in the western Indian Ocean (Zanzibar,Tanzania) were monitored over a 3-year period (2003–2005) and related tocyanobacterial and diazotrophic microbial diversity using a polyphasic approach.Different nitrogenase activity patterns were discerned, with the predominantpattern being high daytime activities combined with low nighttime activities.Analyses of the morphological and 16S rRNA gene diversity among cyanobacteriarevealed filamentous nonheterocystous (Oscillatoriales) and unicellular (Chroo-coccales) representatives to be predominant. Analyses of the nifH gene diversityshowed that the major phylotypes belonged to noncyanobacterial prokaryotes.However, as shown by cyanobacterial selective nifH-denaturing gradient gelelectrophoresis analysis, cyanobacterial nifH gene sequences were present at allsites. Several nifH and 16S rRNA gene phylotypes were related to unculturedcyanobacteria or bacteria of geographically distant habitats, stressing the wide-spread occurrence of still poorly characterized microorganisms in tropical benthicmarine communities.