Oxidative stress of Lake Baikal organisms can be caused by pollution of nearshore with anionic surfactants or heavy metals. Some specific heavy metals are essential for aquatic organisms. At present, there are no data concerning heavy metal content caused by anthropogenic pollution or chemotaxonomic features of Baikal sponges. Here, we consider the applicability of total reflection x‐ray fluorescence (TXRF) to analyze heavy metals in Lubomirskia baikalensis to fill this gap. It was shown that TXRF method can be successfully applied to the fast and reliable quantification of Cu, Zn, Fe, and Mn in this object. Evaluation of different sample treatment procedures showed that the acid digestion by HNO3/H2O2 mixture is an optimal procedure for the preparation of sponges compared with suspension preparation. It helps to improve sensitivity, eliminate the effects of particle size and specimen heterogeneity. The total uncertainty of the TXRF results associated with the sample preparation, taking, depositing, and measuring of the specimen was 6%–18% for Mn, 3%–5% for Cu, 5%–12% for Fe, and 4%–5% for Zn. To validate the developed method, the TXRF results were compared with the data obtained by wavelength dispersive x‐ray fluorescence spectrometry. A good agreement of results was achieved. TXRF data show the dominance of essential heavy metals in L. baikalensis (n = 14) among other heavy metals. Correlations between Cu/Zn contents (R2 = 0.515) as well as between Fe/Mn contents (R2 = 0.972) can be explained by the contribution of them to L. baikalensis antioxidant system.