This study aimed to increase the antifungal and insecticidal activities of NaD1, as an antimicrobial peptides (AMP), by improving its interaction with the fungal cell wall and chitin monomeric units in insect midguts. Hence, the chitin-binding domains (CBDs) of wheat germ agglutinin protein (WGA) were fused to either N- or C-terminus of NaD1 generating transgenic Nicotiana tabacum hairy roots (HRs). Molecular assessments confirmed the integration of NaD1 transgenes, their transcription and production of recombinant peptides in the HR lines. Total protein of (CBD)-NaD1 and NaD1-(CBD) transgenic lines inhibited the growth of Pyricularia oryzae mycelium, suggesting that fusion of CBD to NaD1 can increase NaD1 half-life, leading to higher affinity toward cell wall chitin. Furthermore, feeding the third-instar larvae of Chilo suppressalis with both (CBD)-NaD1 and NaD1-(CBD) extracts exhibited a higher mortality rate. Both NaD1-CBDs caused a significant decrease in trypsin (TRY) and chymotrypsin (CTR) activities in the larvae, while enhancing the activity of antioxidant enzymes CAT, POD, APX, and SOD. Therefore, feeding the larvae by total extract of NaD1-(CBD) and (CBD)-NaD1 HR lines probably increased affinity to midgut chitin in C. suppressalis, enhancing insecticidal activities. Overall, the results indicate that recombinant peptides are effective in enhancing fungal and insect resistance.