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Non-small cell lung cancer (NSCLC) is among the leading causes of human mortality due to a lack of effective treatments. Conventional chemotherapies affect healthy cells and cause multidrug resistance, while tumors may eventually develop resistance to less-toxic targeted therapies. Thus, the need to develop novel therapies for NSCLC is urgent. Here, we show that Nile tilapia-derived Tilapia piscidin (TP) 4 is cytotoxic to a panel of NSCLC cells with different genetic profiles. We observed that TP4 triggers NSCLC cell death through the necrosis and combining TP4 with potent Epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitors (TKI)s, Erlotinib, and Gefitinib, improved drug responses in EGFR-mutated NSCLC cells, but not in EGFR-wild-type NSCLC cells. This work provides novel insights into potential NSCLC treatments, which may utilize antimicrobial peptide TP4 as monotherapy or in combination with EGFR-TKIs.