This experiment aimed to investigate the effects of Pseudomonas growth-promoting bacteria, mycorrhizal fungi, and living mulch on morphological and physiological traits of a medicinal herb, Pimpinella anisum. The study was carried out using a factorial design in a randomized complete block design arrangement with three replications during the crop year of 2019–2020. The first factor includes the use of biological fertilizers (fungi and bacteria) at four levels: 1) control (without the use of biological fertilizers), 2) mycorrhizal fungi, 3) growth-promoting bacteria, 4) combined use of growth-promoting bacteria and fungi. The second factor includes mulch (plant residues) at four levels: 1) control, 2) wheat straw (3 t/ha), 3) berseem clover mulch (2 t/ha), 4) living berseem clover mulch, cultivated among the rows of anise. Cultivation of clover and anise was done simultaneously. Results showed that the combined use of biological fertilizers (Glomus intraradices and Pseudomonas putida) along with living clover mulch led to a significant increase in seed yield, harvest index, and the essential oil yield of anise. Moreover, under such conditions, the highest seed yield (930.8 kg/ha) and the highest essential oil yield (18.15 kg/ha) increased by 48% and 51% compared to the control, respectively. We found that the use of living clover mulch led to a significant increase in biological yield, seed yield, and the number of umbels and umbellets of anise. Accordingly, the highest biological yield of 5332 kg/ha was obtained, which was 55% higher than the control. The extraction of essential oil components showed that α-pinene and para-cymene showed the highest amount and significant components of essential oil. Moreover, the maximum amount of α-pinene (2.277%) observed upon fungi and living clover application along with inter-row clover cultivation (increased by 60% compared to the control), and the maximum value of para-cymene (0.2300%) was obtained upon bacteria and living clover inoculation along with inter-row clover cultivation (increased by 59% compared to the control). Finally, according to the perspectives of sustainable and organic agriculture in medicinal plant cultivation, the use of ecologically compatible inputs such as bacteria and fungi that stabilize food elements, as well as the use of living and non-living plant mulches, can be used as a tool to increase the quantitative and qualitative performance of the anise plant.
