Olive oil, a cornerstone of the Mediterranean diet, is renowned for its high content of phenolic compounds and its protective effects on human health. However, variations in production methods and raw material quality can significantly influence its chemical composition and biological activity. Kara yağ, a traditionally produced, unrefined olive oil unique to Northern Cyprus, is obtained through ancient artisanal extraction techniques and is rich in bioactive phenolics. This study aimed to investigate and compare the effects of Kara yağ and extra virgin olive oil (EVOO) on proliferation, viability, and apoptosis in HCT-116 human colon cancer cells. Samples of Kara yağ and EVOO were obtained from local producers in Northern Cyprus. Their physicochemical parameters, including free acidity, peroxide value, moisture content, and water activity, were analyzed in accordance with the Turkish Food Codex standards. Total phenolic content was determined using the Folin–Ciocalteu method, and antioxidant capacity was assessed spectrophotometrically. The high peroxide value (121.35 meq/kg) of Kara yağ is very high, which means that it was heavily oxidized throughout its conventional manufacturing process, and that heat treatment enhanced the chemical breakdown. Ochratoxin A (2.6 µg/kg) and aflatoxin B1 (0.8 µg/kg) were found in low levels of Kara yağ and therefore, the study is one of the first in the literature to analyze the toxin profile of the Kara yağ. The sensory analysis revealed that EVOO had distinct positive flavor qualities, but Kara yağ did not have them and high concentration of defects. HCT-116 cells were treated with different concentrations (1:1, 1:2, and 1:4) of each oil for 24, 48, and 72 hours. Cell viability was assessed by MTT assay, while morphological and apoptotic changes were evaluated by microscopy and immunocytochemical staining for Ki-67, Caspase-3, and c-Myc. Kara yağ exhibited significantly higher total phenolic content (278.95 mg GAE/kg) and antioxidant activity compared to EVOO. Treatment with Kara yağ led to a marked reduction in cell viability and proliferation in a time- and dose-dependent manner. Enhanced apoptotic activity, characterized by increased caspase-3 expression and decreased Bcl-2 levels, was observed following Kara yağ exposure. Both oils showed comparable c-Myc staining patterns. The findings suggest that Kara yağ, due to its superior phenolic composition and antioxidant potential, exerts stronger cytotoxic and pro-apoptotic effects on HCT-116 colon cancer cells than extra virgin olive oil. These results highlight the potential of traditionally produced Kara yağ as a natural source of bioactive compounds with promising chemopreventive and therapeutic properties against colon cancer. Further studies are warranted to elucidate the underlying molecular mechanisms and confirm its safety and efficacy in vivo.