Broomrape is an obligate root-parasitic weed that acts as a competitive sink for host photoassimilates. Disruption of essential processes for growth of broomrape using host plant-mediated systemic signals can help to implement more specific and effective management plans of this parasite. Accordingly, we tested the possibility of transient silencing three involved genes (PaM6PR, PaCWI, and PaSUS1) in osmoregulation process of broomrape using syringe agroinfiltration of dsRNA constructs in tomato. The highest decrease in mRNA levels, enzyme activity, and amount of total reducing sugars was observed in Phelipanche aegyptiaca when grown on agroinfiltrated tomato plants by PaM6PR dsRNA construct than control. In addition, PaSUS1 dsRNA construct showed high reduction in mRNA abundance (32-fold fewer than control). The lowest decrease in mRNA levels was observed after infiltration of PaCWI dsRNA construct (eightfold fewer than control). While the highest reduction in PaM6PR and PaSUS1 expression levels was detected in the parasite at 3 days post-infiltration (dpi), the maximum reduction in both of the total reducing sugars amount and M6PR and SUS1 activities was observed at 8 dpi. On the contrary, CWI activity, PaCWI expression level, and amount of total reducing sugars in broomrape shoots simultaneously decreased at the day 3 after the dsRNA construct infiltration against PaCWI. On the whole, our results indicated that the three studied genes especially PaM6PR may constitute appropriate targets for the development of transgenic resistance in host plants using silencing strategy.