Cancer stem cells (CSCs) are cells in a malignancy that have the potential to self-renew and differentiate, resulting in a diverse population of cancer cells. These cells are increasingly linked to resistance to traditional therapies, as well as tumour recurrence. Sulforaphane (SFN), a strong anti-cancer and well-tolerated nutritional substance, reduces CSC characteristics and improves gemcitabine therapeutic effectiveness in the non-small cell lung cancer (NSCLC) rat model. Gemcitabine (GEM) and/or SFN enhance these capabilities by reducing transforming growth factor beta1 (TGF-β1) and tumor necrosis factor alpha (TNF-α), which target CD133 and aldehyde dehydrogenase 1A1 (ALDH1A1) CSC markers. This discovery was supported further by changes in histological findings. Furthermore, we demonstrated that GEM and/or SFN had a highly substantial inhibitory effect on the viability of A549 cells. In vitro, combination treatment increased chemotherapeutic drug cytotoxicity. SFN sensitized NSCLC cells to GEM effectiveness, which was accompanied by suppression of GEM-induced CSC formation in lung cancer tissues according to the findings. GEM+SFN, a combination therapy, has demonstrated promising results in restricting the availability of CSCs, suggesting that it might be effective in combating NSCLC resistance and recurrence.