It is increasingly recognized that diffuse, hydrothermal venting is an important source of iron to the deep-sea that can influence oceanic iron dynamics and abundance. Lithotrophic Fe-oxidizing bacteria (FeOB) are dominant at diffuse hydrothermal vent sites, producing microbial iron mats that are often centimeters or more thick. At present, little is known about in situ Fe-oxidation rates, or accretion rates for iron mats. An in situ productivity chamber was developed that took advantage of the unique mineral morphotypes produced by FeOB to estimate rates of Fe-oxidation and accretion. Chambers were placed at two diffuse vents (1179 and 1300 mbsl) at Lo’ihi Seamount. Colonization of the chambers was evident from development of biogenic morphotypes of Fe-oxides. From this analysis, it was estimated that Fe-oxidation rates could range from 8.2–51.9 x 10-6 mol . hr-1, and that iron mats could accrete at around 2.2 cm . yr-1. Molecular analysis indicated that the relative abundance of Zetaproteobacteria, a group of known FeOB, accounted for 80–90% of the bacteria colonizing the chambers. There was a distinct difference between populations at the 1179m site (Pohaku), and the 1300m site (North Hiolo Ridge). Microscope slides placed within the productivity chambers were colonized by recognized and unrecognized morphotypes of FeOB. The cells responsible for one common morphotype that produces a Y-shaped filament were identified as Zetaproteobacteria by use of a small subunit rRNA probe. This work confirms the importance of FeOB in the formation of iron mats, and provides the first estimates for in situ Fe-oxidation rates and mat accretion rates.