Mitophagy selectively removes depolarised and damaged mitochondria, promoting cell viability. Some members of the Orthoflavivirus genus activate mitophagy to allow extended replication times in a surviving cell, while others inhibit mitophagy to favour viral spread. Viral genome replication is catalysed by non-structural protein 5 (NS5), the largest and most conserved protein in the orthoflaviviruses (OFVs). Here we used quantitative proteomics to identify host cell interactors of Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV) NS5 proteins. A total of 141 cellular proteins were found of which 26 were shared in all three datasets. RNAi-mediated knockdown of those factors most significantly enriched revealed putative proviral and restriction factors. Knockdown of host myoferlin and LGALS3BP led to increases in the virus titres, suggesting that both positively promote viral replication. Silencing of mitophagy-related prohibitin 2 (PHB2) led to significant increases in ZIKV and USUV titres, and intracellular levels of genomic RNA, in agreement with an antiviral activity associated with PHB2. Supporting a connection between this restrictive activity and mitophagy, treatment with an inhibitor of this pathway (mdivi-1) resulted in larger virus titres and intracellular genomic RNA. During the course of an infection, PHB2 protein levels gradually decreased, suggesting that it is specifically targeted and degraded by virus-mediated activities. Ectopic expression of NS5 alone did not affect PHB2 intracellular levels in 293T cells, however a significant decrease was detected when NS2B/NS3 protease was expressed. Enhanced PHB2 degradation was observed when both NS2B/NS3 and NS5 proteins were co-expressed, suggesting that NS5 could be acting as a bridge between PHB2 and the viral protease. Overall, our results support that PHB2 is a restriction factor for orthoflaviviral replication which is targeted to degradation to promote viral genome replication.