A field experiment was carried out at the Experimental Farm of El-Kassasin Horticultural Research Station, Agricultural Research Center, Ismailia Governorate, Egypt, to study the effect of partial substitution of chemical nitrogen fertilizer with biofertilizer containing two strains of nitrogen-fixing (Azospirillum lipoferum, Azotobacter chrococcoum), on vegetative growth, flowering and chemical composition of tuberose plants (Polianthes tuberosa L.) under sandy soil conditions to rationalize the use of mineral fertilizers and reduce pollution of the environment in order to increase the quantity and improvement quality of the product on the tuberose plants during the two successive seasons of 2017 and 2018. This experiment included seven treatments with three replications, which were the combinations between two of mineral nitrogen rates (50% and 75%) and three bio-fertilizers of nitrogen-fixing bacteria (Azospirillum lipoferum (S), Azotobacter chrococcoum (Z) and Azospirillum lipoferum + Azotobacter chrococcoum (S*Z) treatments in addition to the control which was 100% N. The treatments were arranged in Randomized Complete Block Design. The results showed that fertilizing tuberose plants with mineral nitrogen at 100% of the recommended dose recorded the highest values for plant height, number of leaves/plant and number of florets/spike. while fertilization with mineral nitrogen at 75% plus the dual inoculation with Azospirillum + Azotobacter  recorded the highest values for fresh and dry weights of leaves (g)/plant, fresh and dry weights of root (g)/plant, fresh and dry weights of spike (g)/plant, floret diameter (cm), spike length (cm), longevity of tuberose cut flower spikes (days), in addition to leaves content of  N, P, K, total chlorophyll a, b and carbohydrates %, as well as essential oil percentage in flowers without significant difference between them and control in most cases in both seasons.