The objective of this study was to determine the effect on methane production of adding different proportions of fresh water spinach to buffalo manure as substrate in in vitro plug-flow biodigesters. The treatments arranged in a completely randomized design with four replicates were ratios of water spinach to buffalo manure of 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45% (DM basis). Measurements were made of gas production, methane content of the gas and pH of the effluent over a period of 30 days from startup of the biodigesters until the end of the experiment. Methane production was increased three times (300%) by including 15% of fresh water spinach in the substrate DM. Water spinach supported intermediate levels of methane production compared with use of household vegetable waste (the best) and fruit waste (the worst) when added to buffalo manure at replacement levels in the range of 15-25% (DM basis).