Aerva sanguinolenta (AS), also called Sambang colok in Indonesian, is a historically utilized herbal treatment in Tawangmangu, Central Java, Indonesia. The herb is claimed to regulate menstrual cycles, inhibit tumor growth, and reduce fever. This study aimed to characterize the phytochemical profile of AS via Liquid Chromatography High-Resolution Mass Spectrometry and to clarify its lethal effects on MCF-7 breast cancer cell lines. The study measured the AS herbal extracts’ antioxidant activity, total phenolic content (TPC), and total flavonoid content (TFC). Solvent extracts (methanol, ethanol, butanol, n-hexane, and ethyl acetate) and liquid-liquid fractions (n-hexane, ethyl acetate, butanol, and water) obtained from methanol extracts were examined. 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide) studies demonstrated considerable cytotoxicity in the n-hexane fraction (IC50 = 49 µg/ml) and ethyl acetate extract (IC50 = 62 µg/ml) against MCF-7 cell lines. Phytochemical investigations revealed the presence of α-Linolenic acid, Galaxolidone, Pinocembrin, and Afzelin in the n-hexane fraction. Extracts demonstrated differing antioxidant (35.53 µg/ml), TFC (14.96 mg QE/g), and TPC (25.61 mg GAE/g) values. In comparison, n-hexane fractions produced unique antioxidant (42.4 µg/ml), TFC [14.96 mg quercetin equivalent (QE)/g], and TPC [25.61 mg gallic acid equivalent (GAE)/g] profiles. N-hexane and ethyl acetate are potential test materials based on cytotoxic test screening against the MCF-7 cell line. N-hexane is the best test material based on all tests conducted, so it becomes the test material that is continued for phytochemical investigations. © 2025 Armansyah Maulana Harahap et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).