Application of bio-fertilizers as plant nutrition is one of the vital solutions to increase plant yield and its quality. Therefore, pot experiment with factorial of completely randomized design with three replications was conducted in the research greenhouse of Zabol University. The first factor was bio-fertilizers which contains two levels of non-inoculated and non-sterile (control) and inoculated soil growing media with bio-fertilizers: Nitrogen fertilizer 1 (microorganism Azeotobacter) and phosphate fertilizer 2 (containing microorganisms Pseudomonas putida, Pantoea agglomrans)  and the second factor  was alfalfa extract from freshly harvested alfalfa and last year harvested alfalfa (old) which was used as foliar and soil application with two levels of concentrations  2 and 4 per thousand with control(total 9 levels). In this experiment, the sunflower (Helianthus annuus L.) was cultivated in non-sterile soil growing media. Pots field capacity was kept at 70% by weighting. The results showed that the highest concentrations of Fe (132.35 mg.kg^-1), Mn (89 mg.kg^-1), chlorophyll a (2.29 mg.g^-1), chlorophyll b (1.07 mg.g^-1) and carotenoids (1.29 mg.g^-1) in sunflower shoot belonged to inoculated growing media with bio-fertilizers and the foliar consumption fresh extracts of alfalfa. The highest potassium concentration was seen in shoot (0.97 percent) in non-inoculated growing media and the foliar consumption fresh extracts of alfalfa was observed at four per thousand level. Resulted also indicated that the maximum dry weight (28.93 g.pot^-1) belonged to non-inoculated with bio fertilizers and fresh foliar application at four per thousand level.