In order to study the effect of irrigation withholding and biofertilizers application on quantitative and qualitative yield and grain filling duration of sunflower (Helianthus annus L.), a factorial experiment was conducted based on randomized complete block design with three replications in research field of Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Iran in 2015. Treatments were four biofertilizers levels [(no biofertilizer, seed inoculation by Azotobacter chrocoocum strain 5, application of mycorrhiza (Glomus mosseae), Azotobacter chrocoocum + Glomus mosseae) and irrigation in three levels (full irrigation as control; moderate water stress or irrigation withholding at grain filling period; severe water stress or irrigation withholding at flowering stage]. Results showed that number of unfilled and filled grains per head, grain filling rate, 1000- grain weight, oil percentage and chlorophyll index, grain filling duration and effective grain filling duration were significantly affected by irrigation levels and biofertilizers. The highest grain filling rate (1.95 g per day), grain filling duration (21.18 days), and effective grain filling duration (35.85 days) were obtained in full irrigation and application of Azotobacter + mycorrhiza. Irrigation withholding at flowering stage led to 26, 5.18, 42.48 and 16.88% decreases in number of filled grains per head, 1000-grain weight and chlorophyll index and oil percentage, respectively, in comparison with full irrigation. Whereas, application of Azotobacter + mycorrhiza led to 15.24, 3.48, 30.16 and 9.25% increases in number of filled grains per head, 1000-grain weight, chlorophyll index and oil percentage, respectively, in comparison with control (no biofertilizers). Under severe water stress, application of biofertilizer (Azotobacter + Glomus) led to 13.3% increase in grain yield in comparison with lack of biofertilizers. Based on the results, it seems that application of biofertilizers is suitable for increasing sunflower yield under water stress condition.