Safflower is an important industrial oil seed and bioenergy crop in semi-arid subtropical regions due to its potential to grow on marginal land and having good percentage of seed oil contents which is an important parameter for biofuel production. However, it is an ignored crop in Pakistan. In order to improve the crop productivity and reduce the use of agrochemicals for sustainable biodiesel feedstock production, an experiment was conducted for two years to improve the fatty acid composition and oil quality of Carthamus tinctorius L. (safflower) by the inoculation of Azospirillum and Azotobacter alone as well as in combined application with nitrogen and phosphate (NP) fertilizers on cultivars Thori and Saif-32 under field conditions. Separation and quantification of fatty acids were done on precise comprehensive two-dimensional gas chromatography (GC×GC). The results showed that fatty acid profile specifically monounsaturated fatty acids i-e oleic acid (C18:1) was significantly improved by Azospirillum supplemented with the quarter dose of NP fertilizers (SPQ) with concomitant decrease in polyunsaturated fatty acids by the respective treatment. Oil quality attributes such as acid value, saponification number, iodine value, refractive index and free fatty acid contents were reduced by the application of Azotobacter and Azospirillum in combination with half and quarter doses of NP fertilizers treatments (BTH, SPH, BTQ and SPQ). The reduction in these variables is positively linked with improved biodiesel yield and quality. It can be concluded that application of Azospirillum and Azotobacter not only reduced the use of NP fertilizers up to 50%-75% but also improved the oil quality in order to obtain environment friendly, sustainable and green fuel.