As hepatitis C virus (HCV) is one of the major health problems in many countries, interest has been aroused in the design, synthesis, and optimization of novel NS5A inhibitors, outside the chemical space of currently available direct acting antivirals (DAAs). Two series of symmetric molecules with core scaffold 3,3′-(buta-1,3-diyne-1,4-diyl)dianiline or 4,4′-(buta-1,3-diyne-1,4-diyl)dianiline, coupled on its nitrogen as amide with different end caps, were synthesized and tested for their activities against HCV by using cell-based antiviral assays. Molecules with the 3,3′-(buta-1,3-diyne-1,4-diyl)dianiline core were more active than their 4,4′-congeners. Only the 3,3′-derivatives showed noncoplanarity of core phenyls that mostly led to a better interaction with the target protein and appears to be a crucial element for efficient inhibition of HCV replication. Compounds 2f and 2q exhibited potent inhibition of genotype (GT) 1b HCV replication with EC 50 values in the picomolar range and selectivity index greater than 6 orders of magnitude. The compounds seem more selective toward GT 1b and 4a . In conclusion, novel symmetric molecules with a 3,3′-(buta-1,3-diyne-1,4-diyl)dianiline core are potent and selective inhibitors that provide new extension to explore the structure–activity relationship of NS5A targeting DAAs.