Iron fabricated bio-digesters A and B (32L each) were used to investigate microbial potency of biogas production from co-digested water hyacinth and poultry litter (5.76kg) mixed in the ratio of 1:5, using 0.4kg wood ash treatment for digester A and 18L of water for each digester. The digesters contents were stirred daily to ensure homogenous dispersion of constituents. Gas production, measured in litters was obtained by water displacement. Total microbial count, ambient and slurry temperatures, slurry pH, volume of gas produced and gas flammability readings were taken. Correlations, regression and t-test analyses were used at p=0.05. Results showed that digester A produced more gas over the retention time (32days), and had an earlier onset of flammability (4 days) compared with digester B. both digesters had temperature range of 28_0C?36_0C. Ambient temperatures were lower than that of the digesters (28_0C?34_0C). Both digesters started with a slurry pH of about 6, which then dropped to 4, rose to 7, then sustained slightly below 6 for digester B, and slightly above 6 for digester A. Biogas production and total microbial count for digester A had a strong positive correlation, while those of digester B had a weak negative correlation. T-test analysis revealed significant difference (p<0.05) in biogas production, total microbial count and pH variation between the digesters. Gas composition analysis for both digesters showed methane concentrations to be above 60%. Pathogenic bacteria like Staphylococcus spp., Klesiella spp., E. coli and Salmonella spp. were isolated from both digesters. It was concluded that wood ash treatment significantly increased biogas production and enhanced the biogas production process from co-digested water hyacinth and poultry litter.