The possibility of introduction of foodborne indicator bacteria into iru by processing-water samples used for washing boiled seeds cotyledons prior to traditional fermentation of iru was investigated. Thirty processing water samples (collected from 30 locations in five major cities of southwestern and middle belt Nigeria (Abeokuta, Ibadan, Ijebu-Ode, Lagos and Lokoja) were total coliform positive between 1 month and two years of storage, while 27 (90.0%) and 23 (76.7%) of the water samples were total coliform positive at 28 and 35 months of storage, respectively. Using standard phenotypic taxonomic tools, the viable and easily recoverable Gram-negative bacterial isolates, obtained from the water samples were identified as- Enterobacter aerogenes (5.8%), Escherichia coli (58.9%), Klebsiella aerogenes (11.8%), Klebsiella pneumoniae (11.8%), Proteus mirabilis (5.8%) and Pseudomonas aeruginosa (5.8%). The overall bacterial isolates from various simulated, fermenting iru samples were characterised as Enterobacter aerogenes (11.2%), Escherichia coli (22.3%), Klebsiella aerogenes (8.6%), Klebsiella pneumoniae (24.3%), Morganella morganii (3.3%), Salmonella Enterica serovar Paratyphi (5.3%), Salmonella Enterica serovar Typhi 4 (2.6%), Shigella dysentariae (4.6%), Proteus mirabilis (13.8%), Pseudomonas aeruginosa 4 (2.6%) and V. cholerae (1.3%) but the most recovered bacterial species from the fermenting mesh in the first 5 days of simulated fermentation were Enterobacter aerogenes, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae and Proteus, mirabilis, while controlled iru fermentations indicated that the fermenting bean cotyledons washed with processing-water samples had higher recovery rates of indicator bacteria than the fermenting cotyledons washed with tap and sterile water samples. The study concluded that water used in the traditional production of iru can be source of foodborne indicator bacteria.