Barleria albostellata C.B. Clarke (Acanthaceae) is a plant native to South Africa and relatively few studies have been performed on it. Species in this genus are known for their ethnopharmacological and phyto-medicinal values. In this study, the total flavonoid and phenolic contents and the antioxidant and cytotoxic activities of hexane, chloroform, and methanol extracts were evaluated at five different concentrations (15, 30, 60, 120, and 240 μg/mL). The antioxidant activity of the extracts of B. albostellata was assessed in vitro using the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging and ferric reducing antioxidant power (FRAP) assays, while the phenolic content was determined using a Folin–Ciocalteu assay. The extracts’ cytotoxicity was established using a 3-[(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay in human embryonic kidney (HEK293), cervical cancer (HeLa), and breast adenocarcinoma (MCF-7) cell lines. Methanolic leaf extracts had the highest total flavonoid content (42.39 ± 1.14 mg GAE/g DW) compared to other solvents. Additionally, the total phenolic content was the greatest in the methanol leaf extract (6.05 ± 0.09 GAE/g DW), followed by the methanol stem extracts (2.93 ± 0.73 GAE/g DW). The methanolic leaf and stem extract concentrations needed for 50% inhibition (DPPH) were 16.95 µg/mL and 14.27 µg/mL, respectively, whereas for FRAP, the reducing powers of all extracts were considerably lower than the ascorbic acid standard. The IC50 values of extracts tested in the three cell lines were >63 µg/mL. According to the findings of our study, the leaves and stems of B. albostellata are rich in several bioactive compounds that may be a possible source of natural antioxidants and may have the potential to treat certain diseases. The extraction of the bioactive compounds from the leaves and stems of B. albostellata using bioassay-guided fractionation and the assessment of their safety will be essential for further investigations into this species in the search of potential novel therapeutic drug leads. To the best of our knowledge, this is the first report of the cytotoxic activities of leaf and stem extracts of Barleria albostellata.