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Background: Autophagy is reported as a survival or death-promoting pathway that is highly debatable in different kinds of cancer. Here, we examined the co-effect of cold atmospheric plasma (CAP) and silymarin nanoemulsion (SN) treatment on G-361 human melanoma cells via autophagy induction. Methods: The temperature and pH of the media, along with the cell number, were evaluated. The intracellular glucose level and <i>PI3K/mTOR</i> and <i>EGFR</i> downstream pathways were assessed. Autophagy-related genes, related transcriptional factors, and autophagy induction were estimated using confocal microscopy, flow cytometry, and ELISA. Results: CAP treatment increased the temperature and pH of the media, while its combination with SN resulted in a decrease in intracellular ATP with the downregulation of <i>PI3K/AKT/mTOR</i> survival and <i>RAS/MEK</i> transcriptional pathways. Co-treatment blocked downstream paths of survival pathways and reduced <i>PI3K</i> (2 times), <i>mTOR</i> (10 times), <i>EGFR</i> (5 times), <i>HRAS</i> (5 times), and <i>MEK</i> (10 times). CAP and SN co-treated treatment modulates transcriptional factor expressions (<i>ZKSCAN3</i>, <i>TFEB</i>, <i>FOXO1</i>, <i>CRTC2</i>, and <i>CREBBP</i>) and specific genes (<i>BECN-1</i>, <i>AMBRA-1</i>, <i>MAP1LC3A,</i> and <i>SQSTM</i>) related to autophagy induction. Conclusion: CAP and SN together activate autophagy in G-361 cells by activating <i>PI3K/mTOR</i> and <i>EGFR</i> pathways, expressing autophagy-related transcription factors and genes.