Newcastle disease (ND), caused by Orthoavulavirus javaense (OAVJ), is a significant threat to the poultry industry and outbreaks of virulent strains can lead to substantial economic losses. Studies to identify molecular pathways that can be used for intervention or to reduce pathology are critical for mitigating losses due to ND. In this study, we showed that chicken miR-26a-5p can inhibit the replication of both the lentogenic and velogenic pathotypes of OAVJ by acting via two distinct mechanisms, namely, by regulation of the host interferon response and by direct targeting of the viral polymerase gene. Mir-26a-5p upregulation inhibited the replication of both lentogenic and velogenic OAVJ strains. Stable overexpression of miR-26a-5p led to the down-regulation of multiple genes in the innate immune sensing pathway and led to a small but significant increase in viral titer for a velogenic OAVJ strain suggesting a pro-viral role. However, this study also identified a site for the direct binding of mir-26a-5p to the OAVJ polymerase gene which is highly conserved across the majority of class II strains. Furthermore, transfection of the miR-26a-5p mimic following viral infection demonstrated a direct inhibition of polymerase transcripts while inhibitor transfection led to partial rescue of the miR-26a-5p mediated repression. These data identified new roles for chicken miR-26a-5p in regulating OAVJ replication.