MicroRNA-34a (miR-34a) is recognized for its role as a tumor suppressor, influencing critical cellular processes such as cell cycle arrest, apoptosis, and senescence. Its dysregulation is linked to various cancers, making it a potential target for therapeutic intervention. This study investigates the cytotoxic effects of miR-34a in three human cancer cell lines: colorectal adenocarcinoma (Caco2), non-small cell lung cancer (NSLC), and hepatocellular carcinoma (HepG2). The research involved transfecting these cell lines with miR-34a mimic and inhibitor, followed by a 48-hour incubation and assessment of cytotoxicity through cell proliferation assays. Additionally, Notch1 gene expression was evaluated using Syber-green-based Real-time quantitative PCR (qPCR). Results showed that miR-34a mimic significantly reduced cell viability in all three cancer cell lines, with a 30% decrease in Caco2 cells. Conversely, the miR-34a inhibitor increased cell viability, with Caco2, A549, and HepG2 cells showing 16%, 11%, and 14% increases, respectively. The Notch1 gene expression was notably decreased with miR-34a mimic—an 8-fold reduction in Caco2, a 4-fold reduction in A549, and a 6-fold reduction in HepG2 cells. In contrast, miR-34a inhibitor led to a significant increase in Notch1 expression. These findings highlight miR-34a's potent cytotoxic and anti-proliferative effects through the downregulation of the Notch1 signaling pathway, suggesting its potential as a therapeutic target in cancer treatment.