Background: Colorectal cancer is a leading cause of cancer-related mortality worldwide, underscoring the need for effective therapies with lower toxicity. Probiotic-derived metabolites (postbiotics) have emerged as promising anticancer components with immunomodulatory and antiproliferative effects. Methods: Cell-Free Supernatant (CFS) from Results: Growth under microaerophilic conditions yielded a sterile CFS that was subsequently profiled by GC-MS. Cytotoxicity was assessed by MTT assay, while apoptosis and cell-cycle distribution were evaluated by flow cytometry. GC-MS showed more than 65 chromatographic peaks, and 28 major compounds were tentatively identified by NIST-library matching; diketopiperazines accounted for approximately 45% of the total identified peak area. The study-strain CFS reduced HT-29 cell viability in a concentration-dependent manner, with an estimated IC50 of 14.2 micrograms/mL expressed as total lyophilized solids. Annexin V-FITC/PI flow cytometry showed 47.6 ± 2.9% early apoptotic, 2.3 ± 0.8% late apoptotic, 2.3 ± 0.6% necrotic, and 47.8 ± 3.2% viable cells after treatment. Treated cells were distributed as 47.3% G1, 38.9% S, 3.22% G2/M, and 2.60% sub-G1; because reliable quantitative control data were unavailable, phase-specific arrest could not be established. Conclusion: The study-strain CFS showed concentration-dependent in vitro cytotoxic and pro-apoptotic activity against HT-29 cells. The activity may be associated with the partial metabolite profile identified by GC-MS, but specific active compounds and molecular pathways were not established. The cell-cycle findings are descriptive because reliable quantitative control data were unavailable. Further studies using processed-medium controls, non-transformed colonic cells, additional cancer cell lines, orthogonal viability assays, mechanistic markers, and activity-guided fractionation are required.