This study investigates the glucosinolate profiles of various Capparis spp. and Maerua baillonii, from the Capparaceae family, and evaluates the cytotoxic potential of volatile isolates from Capparis spinosa subsp. rupestris. Using UHPLC‐DAD‐MS/MS, GSLs were identified and quantified across different plant tissues. Glucocapparin, predominant glucosinolate in C. spinosa subsp. spinosa, C. spinosa subsp. rupestris, and M. baillonii, was isolated and analyzed by NMR in its desulfo‐form. Notably, glucosinolates were absent in C. richardii and specific tissues of M. baillonii (e.g., leaves and stems), underscoring species and/or tissue‐specific variability within Capparaceae. Less common glucosinolates for Capparaceae plants, such as glucocochlearin, glucohirsutin, and glucoarabin were also detected. Methyl isothiocyanate was found to be the main volatile in all isolates obtained through various isolation methods, comprising ca. 80‐90% of the total volatiles. Methyl isothiocyanate‐rich volatile isolates were tested for cytotoxicity against human breast cancer (MDA‐MB‐231) and bladder cancer (T24) cell lines using the MTT assay, showing significant activity with IC50 values of 3.81 µg/mL and 5.95 µg/mL, respectively. These findings underscore the anticancer potential of glucocapparin‐bearing plants from Capparaceae family.