Abstract: Root parasitic weed Orobanche aegyptiaca adversely affects yield and quality of tomato (Solanum lycopersicum). The means of effective control is still the focus in O. aegyptiaca research. Fungus is one of the biocontrol agents of Orobanche spp.. However, few studies have been done on the use of non-pathogentic fungi to control Orobanche spp. weed. In this study, the effects of cell-free culture filtrate of Penicillium griseofulvum, a non-pathogentic fungus strain of O. aegyptiaca, on O. aegyptiaca seed germination and germ tube growth were investigated in a petri-dish experiment. In addition, a pot experiment was conducted to explore the effect of powdered P. griseofulvum inoculum on weedy O. aegyptiaca control. The effects of P. griseofulvum inoculum on the growth of host tomato plants and the change in microflora in rhizosphere soils of tomato plants were also investigated. Results showed that:1) in the petri-dish experiment, cell-free culture filtrate of P. griseofulvum significantly inhibited both O. aegyptiaca seed germination and germ tube growth. When O. aegyptiaca seeds and tomato seedlings were co-cultured for 6 days, O. aegyptiaca seed germination was completely inhibited (100.0%) in treatments with P. griseofulvum cell-free culture filtrate. After co-culturing for 6 days, O. aegyptiaca seed germination rates reduced by 80.26%, 70.26%, 68.10% and 47.51%, respectively in treatments with volume ratios of P. griseofulvum cell-free culture filtrate and Hogland nutrient solution ratios of 1:2, 1:4, 1:6 and 1:8. The lengths of O. aegyptiaca germ tubes significantly reduced by 100.00%, 68.84% and 19.24%, respectively when treated by undiluted, 10-fold diluted and 100-fold diluted P. griseofulvum cell-free culture filtrate. 2) In the pot experiment, P. griseofulvum inoculum inhibited the emergence of O. aegyptiaca tubercles and the growth of individual O. aegyptiaca tubercle, but simultaneously increased tomato fruit yield. The number of epigeal O. aegyptiaca tubercles, epigeal rate of O. aegyptiaca tubercles and dry weight of individual O. aegyptiaca tubercles all significantly reduced after the application of powdered P. griseofulvum inoculum at 1.0 g·kg^-1 for 130 days respectively by 76.19%, 85.30% and 28.48% than the control. After the application of P. griseofulvum inoculum for 130 days, tomato fruit yield was 346.8 g per plant (51.57%) more than the control (228.8 g). In addition, P. griseofulvum also adjusted microflora structure in rhizosphere soils of tomato plants. The application of P. griseofulvum inoculum reduced fungi population (excluding CF3) and significantly increased population ratio of bacteria to fungi in rhizosphere soils of tomato plants by 75.60% and 117.57%, respectively, compared with the control. On average, application of P. griseofulvum inoculant reduced fungi population (excluding P. griseofulvum) and increased actinomycetes population in rhizosphere soils of tomato plants respectively by 42.81% and 84.15% over the control. In conclusion, P. griseofulvum had the ability to control O. aegyptiaca infection of tomato plant with fungus as suitable biological agent to control O. aegyptiaca.