In this research, the study utilized the root, leaf, and petiole parts of in vitro grown Salvia hispanica plants as explants. Following UV-C treatment applied to developing callus, methanol extracts were obtained and analyzed using liquid chromatography– mass spectrometry (LC/MS) to investigate their anticancer properties. First, the seeds of S. hispanica were soaked in commercial bleach for 6 min to ensure surface steriliza- tion. The most effective antimicrobial activity on Gram-negative bacteria, with a zone diameter (11 ± 0.82 mm), was noticed in callus extracts obtained from the petiole explant in the second protocol against Klebsiella pneumoniae EMCS bacteria. Antican- cer activities on SH-SY5Y human neuroblastoma cells were investigated by using 1000, 500, 250, 125, 62.5, 31.25, 15.62, and 78.12 μg/mL doses of the extracts, and the most effective cytotoxic activity was determined at the 1000 μg/mL dose of the extracts obtained from both protocols. The extracts were determined to inhibit hCAI, hCAII, AChE, and BChE enzymes. The content of 53 different phytochemical compo- nents of the extracts was analyzed by liquid chromatography with tandem mass spec- trometry (LC–MS/MS). Rosmarinic acid, quinic acid, and caffeic acid were found in the highest concentration. The comprehensive LC–MS/MS analysis of S. hispanica extracts revealed a diverse array of phytochemical compounds, highlighting its poten- tial for therapeutic applications.