Background: Cancer is a leading cause of mortality, challenges in conventional nonsurgical cancer treatments, such as chemotherapy and radiation and include poor prognosis, recurrence, and low survival rates. Objective: The primary objective of this study was to investigate the potential antitumor activity of the venom extracted from an Egyptian spider, preliminarily identified as Dysdera sp. The focus is on exploring the cytotoxicity of the spider venom against various tumor cell lines, including lung (A549), hepatocellular (HEPG-2), colon (HTC-116), and breast (MCF-7) cells, in comparison with non-tumorigenic WI-38 cells. Material and methods: The Egyptian spider was hunted from Fayoum governorate, and its venom was extracted in PBS for further investigation. MTT assay was employed to assess the potential cytotoxicity of the venom against the specified tumor cell lines. The study aims to determine the IC50 values, representing the concentration of the venom at which 50% inhibition of cell growth occurs. Results: The results of the MTT assay revealed varying IC50 values, ranging from 163 to more than 1000 µg/ml, against the different tumor cell lines. Notably, the spider venom exhibited promising antitumor activity against HEPG-2 and A549 cells, with IC50 values of 163 and 432 µg/ml, respectively. These findings suggest that the crude venom may contain elements that could be explored for their potential as anticancer medications. Conclusion: This study demonstrated the potential antitumor activity of the Dysdera spider venom, particularly against HEPG-2 and A549 cells. The varying IC50 values indicated the specificity of the venom's cytotoxic effects on different tumor cell lines.