This study was conducted to investigate the effect of biomarinading using different starter cultures on tenderness of tough buffalo's meat. 3x3 cm uniform-sized from Semitendinosus muscle of over 5 years female buffalo's meat "Bubalus bubalis" were biomarinaded with a mixture of Lactococcus lactis subsp. cremoris and L. lactis subsp. Lactis, and with Lactobacillus helveticus at a level of 3.00 log10 CFU/g and kept at 22°C for 17 hours; 40°C for 6 hours, respectively then cooked by three different cooking methods (water bath, hot air oven and microwave). The samples were subjected to sensory, bacteriological, physico-chemical, electrophoretic and histological evaluations. The results of sensory evaluation showed that L. helveticus significantly improved (P< 0.05) the overall acceptability of samples cooked by dry heat, whereas L. cremoris improved the overall acceptability of chunks cooked by heating radiation. Physico-chemical parameters showed significant increase myofibrillar fragmentation index, sarcomere length and significant decrease muscle fiber diameter while, none significant changes in protein solubility, collagen content, collagen solubility% and shear force values. Electrophoretic pattern of muscle proteins also revealed extensive proteolysis and reduction in number of protein bands. While, histological evaluation showed significant changes in the ultrastructure of muscle fiber in raw and cooked samples with little changes of connective tissue.