The Paper describes the comprehensive experimental work carried out on M40 grade bacterial concrete containing fly ash. Two types of common soil bacteria namely, Bacillus Pasteruii and Bacillus Odysseyi with a concentration of 105 cells/ml have been used. The optimum cell concentration of bacteria was arrived at by studying its influence on compressive strength of cement mortar matrix. Fly ash of 10 % by weight of cement was used to partially replace OPC in the concrete mixture. The performance of M40 bacterial concrete containing fly ash was assessed by testing the standard specimen for compressive strength, flexural strength and split tensile strength at different ages of curing. Results of investigation indicate significant improvement in 28 days compressive strength in the range of 12% to 27%. Twenty eight days flexural strength and split tensile strength have also shown appreciable increase in the range of 4% to 14% and 11% to 24% respectively. In general, the results of investigation are encouraging and set in positive direction for use of Bacterial concrete in the construction sector during 21st century.