Vitamin B12, or cobalamin, is an essential human vitamin and most commonly acquired in the diet through consumption of animal products. Acquisition of the vitamin for people who follow strict plant-based diets is limited to supplementation via tablets or consumption of fortified foods. Tempeh is an Indonesian food of soybeans fermented with a filamentous fungi, Rhizopus sp. is a potential dietary source of cobalamin, but its presence is under question based on difficult quantification of this trace vitamin. This study tested the presence and concentration of vitamin B12 in commercially available tempeh and tempeh produced with deliberate inoculation of various bacteria and moulds. Vitamin B12 was not detected in any commercially available tempeh. Tempeh made with filamentous fungi and Propionibacterium freudenreichii (ATCC 9617) consistently produced detectable levels of cobalamin. P. freudenreichii co-fermented with Rhizopus oligosporus (ATCC 22959) produced the highest concentration of cobalamin at 8.26 +/- 0.13 micro g/100g of wet weight of tempeh. Co-fermentation with a different tempeh mould significantly reduced the amount of cobalamin (P = 0.008). Results from this study suggest that incorporating nutrient-enhancing microbes into an existing fermentation stage of a product is an effective method to increase the nutritional density of food. The results of this study suggest that cheap, safe and easily cultured microbes can contribute to the nutritional diversity of people following plant-only diets.