Introduction: Bioactive compounds from natural sources have gained attention for their diverse biological activities. Marine macroalgae are rich in secondary metabolites with potential pharmacological value. Evaluating their cytotoxic effects on neuroblastoma cell lines may aid in identifying novel candidates for anticancer research. Specifically, Padina gymnospora (PDG) has been identified as a potentially valuable source of bioactive compounds that may possess cytotoxic characteristics. Methods: The extraction of PDG was performed, followed by preliminary phytochemical evaluation, Fourier-transform infrared spectroscopy (FTIR), and high-resolution liquid chromatographymass spectrometry (HR-LCMS QTOF) analyses. The cytotoxic effects were assessed utilizing the MTT assay on SH-SY5Y neuroblastoma cell lines. Results: The phytochemical analysis indicated the presence of tannins, phenolic compounds, alkaloids, cardiac glycosides, steroids, carbohydrates, inorganic elements, as well as vitamins A and C. The HR-LCMS investigation identified 25 compounds in the positive ionization mode and 10 compounds in the negative ionization mode. The MTT assay revealed substantial cytotoxic activity, yielding an IC50 value of 62 µg/ml. Discussion: The diverse range of metabolites identified in the PDG extract suggests a potential synergistic interaction contributing to the observed cytotoxic activity. The presence of multiple bioactive constituents highlights the pharmacological potential of seaweed-derived compounds. These findings warrant further investigation to determine the individual roles of these metabolites and to elucidate their underlying mechanisms of action. Conclusion: The PDG extract exhibited measurable cytotoxic activity against neuroblastoma cells, with an IC50 value of 62 µg/ml, indicating moderate inhibitory potential. These findings highlight the presence of bioactive constituents within the extract and suggest that Padina gymnospora may serve as a promising source of compounds for further cytotoxic and pharmacological investigations.