Endocrine disrupting chemicals (EDCs) are of increasing concern due to the ecotoxicological risk they pose to marine organisms. Bisphenols, parabens, and triclosan are especially noteworthy for their toxic and long-lasting impact on the environment. The present research evaluates the presence and bioaccumulation of 20 EDCs (12 bisphenol homologues, 7 parabens and triclosan) in the body wall of Holothuria tubulosa Gmelin 1791 specimens by using gas chromatography-tandem mass spectrometry. This part of the sea cucumber is used by humans as food, mainly in Asian Eastern countries. The key variables in ultrasound-assisted compound extraction and chromatographic processes were optimized. The method was validated, obtaining acceptable analytical parameters according to the used guide. The linearity of the method was statistically validated (R2 > 99% and Plof > 5% in all cases). The limits of detection were between 0.1 and 0.7 ng g−1 and the limits of quantification were between 0.5 and 2 ng g−1. For the determination of the accuracy (trueness and precision) of the method, a recovery test was performed. Recovery rates ranged from 85.6% to 112.3%, and precision (in terms of standard deviation) was less than 15% in all cases. Specimens of Holothuria tubulosa Gmelin 1791 collected from 7 sampling areas on the Southern Coast of Spain were analyzed. The results revealed the presence of multiple compounds in all samples, with bisphenol A being the predominant bisphenol (100% of the samples) and methylparaben the most abundant paraben. Highest concentrations were found in areas with greater anthropogenic activity, indicating a situational trend. This study underscores the value of Holothuria tubulosa as a bioindicator of marine contamination, raising awareness about the presence and harmful effects of EDCs on marine life and emphasizing the need for conservation and protection strategies for aquatic ecosystems.