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 sterilization. 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. Anticancer 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 components of the extracts was analyzed by liquid chromatography with tandem mass spectrometry (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 potential for therapeutic applications.