Microalgae is a rich source of polyunsaturated fatty acid. This study was conducted to identify and isolate microalgal strain with the potentials for producing polyunsaturated fatty acids (PUFAs) and determine its cytotoxic effect on some cancer cells. The algal strain (Chlorella sp. S14) was cultivated using modified BG-11 media, and algal biomass obtained was used for fatty acid extraction. Gas chromatographic–mass spectrometry was used to identify and quantify the levels of the fatty acid constituents. The total content of monounsaturated fatty acids (1.12%) was low compared to polyunsaturated fatty acids (PUFAs) (52.87%). Furthermore, n-3 PUFAs accounted for (12.37%) of total PUFAs with the presence of α-linolenic acid (2.16%) and cis-11,14,17-eicosatrienoic acid (2.16%). The PUFA-rich extract did not exhibit a cytotoxic effect on normal cells. Treatment with the PUFA-rich extract (150 µg/mL) significantly reduced cell viability in MCF-7 (31.58%) and A549 (62.56%) cells after the 48 h treatment. Furthermore, treatment of MCF-7 with fatty acid extracts (125 and 150 µg/mL) showed a significant reduction in MDA levels, increase in catalase activities and decrease in GSH level compared to untreated cells. However, a slight decrease in MDA level was observed in A549 cells after the 48 h treatment. There are no significant changes in catalase activities and GSH level in treated A549 cells. However, a slight reduction of NO levels was observed in treated MCF-7 and A549 cells. These results indicate the potentials of PUFA-rich extracts from Chlorella sp. S14 to reduce viability and modulate redox status in A549 and MCF-7 cells.