Kinetics and mechanism of oxidation of two biologically reactive heterocyclic compounds, namely, barbituric (BA) and thiobarbituric (TBA) acids by chromium trioxide (CrVI) have been explored using UV–Vis absorption spectra in sulfuric and perchloric acid solutions at fixed ionic strength of 2.0 mol dm−3 and a temperature of 298 K. The oxidation reactions demonstrated a 3:4 stoichiometry (organic acid: CrVI). The reactions kinetics in the examined acidic media were set to be first order with admiration to [CrVI] and fractional orders in organic acid and H+ concentrations. Varying the ionic strengths and dielectric constants of the reaction media had no noteworthy influence on the reactions’ rates. Tests for free radicals involvement throughout the reactions were positive. Addition of chromium(III) as a predicted oxidation product did not considerably alter the rates whereas addition of manganese(II) diminished the oxidation rates. Supplementation of some divalent transition metal ions increased the oxidation rates. At same investigational circumstance, the rates of BA oxidation in both acidic media were set to be about 20–30% higher than those of TBA, and those acquired in sulfuric acid were also found to be higher than those of perchloric acid solutions for both BA and TBA with almost the same percentages. The oxidation products of BA and TBA were described as 2,4,5,6-pyrimidinetetraone (alloxan) and 2-thio-4,5,6-pyrimidinetrione, respectively. A conceivable oxidation mechanism has been anticipated. The derived rate-law expression was in a good agreement with the investigational outcomes. The activation parameters with regard to the second order rate constants were assessed and debated. The existing study presents an unprecedented simple and inexpensive treatment method for removal of certain pollutants for saving the environment and human health.