378-383Rhizobium forms symbiotic relationship with leguminous crops and is recommended for use in various legumes. Rhizobium sp. fix atmospheric nitrogen and make it available to legumes through formation of root nodules. Rhizobium biofertilizer production is carried out mostly by using semi-synthetic microbiological medium which forms major expense of this activity. Successful commercial production of biofertilizer can be enhanced by use of natural substrates, as molasses, cheese whey, corn steep liquor, for bacterial biomass production. The present work centers around the use of sugarcane molasses as a source of fermentable sugars. It was supplemented with various organic/inorganic nitrogen sources, chemical compounds to increase biomass yield and to increase the shelf life of the product thus prepared. Compliance to Fertilizer Control Order specifications was demonstrated in wet lab analysis