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Fennel fruits (Foeniculum vulgare Mill.) represent one of the plant-based natural spices. This study aims to improve the fruit yield and essential oil (EO) quality by reducing the undesirable component, estragole, under different fertilizer treatments. The fertilizers included chemical nitrogen, phosphorus, and potassium (NPK), and also the organic additive, rabbit manure (RM). For bio-fertilization, plants were inoculated with a mixture of N-fixing bacteria, and P- and K-solubilizing bacteria with/without vesicular-arbuscular mycorrhizal fungi. The results showed that fruit and EO yield parameters, total phenolic content (TPC), total flavonoid content (TFC), and DPPH scavenging activity of fruit extracts were enhanced by fertilizer treatments in both growing seasons. NPK at 150% of the recommended dose (NPK150) and RM at 60 m3/fed (RM60) recorded the highest values of plant height, umbel number/plant, 100-fruit weight, fruit yield, chlorophyll, carbohydrates, N and P content, EO content, and yield. TPC and TFC were enhanced by using biological fertilizers. DPPH scavenging activity was higher in organically and biologically fertilized fennel. The GC-MS analyses of EO revealed higher contents of the desirable trans-anethole in the organically and chemically fertilized fennel. However, the highest proportion of estragole, the undesirable compound, was recorded for NPK150 and unfertilized plants. On the contrary, increases in the EO content and yield of fennel fruits were achieved by RM along with a reduction in estragole, enhancement in trans-anethole, and increments in other favorable compounds such as fenchone and limonene. In addition, the inhibition of estragole formation was recorded with bio-fertilizers, which also increased the trans-anethole content. Furthermore, the trans-anethole/estragole ratio was significantly higher with the application of organic and bio-fertilization. Hence, organic and bio-fertilizer resources can produce high-quality fennel fruit and EO. The reduction in the use of chemical fertilizers can help to reduce environmental pollution.