This study introduces a novel group of acridinium salts, specifically 10-methyl-9-((phenylthio)carbonyl)acridin-10-ium trifluoromethanesulphonates (ATEs), which are the sulphur analogues of widely used acridinium esters in bioanalytics. We conducted comprehensive experimental and theoretical studies on the chemiluminescence (CL) properties of these ATEs, including emission efficiency and kinetics in various liquid environments. These findings provide insights into the unique chemiluminescence behaviour of ATEs and pave the way for their potential applications in luminometric assays. All ATE compounds, regardless of the type and location of the substituent, exhibit an effective flash-type emission. The 2-methoxy and 4-nitro-substituted derivatives demonstrated the highest effectiveness in an organic environment (dimethylsulfoxide, ethanol). We further evaluated the analytical usefulness of selected ATEs by studying their interactions with biological antioxidants (AOs) in aqueous systems. These interactions were found to reduce the emission capability of CL in proportion to the AO concentration in the system. The linearity ranges of AO analyses were within the range 10−9–10−6 M, with detection limits at the level of 10−7 M. Quantum chemical (DFT) calculations on the thermodynamics and kinetics of ATE chemiluminescence disclosed that they are highly susceptible to nucleophilic addition of the OOH− ion, initiating the CL process. They also demonstrated the thermodynamic feasibility of producing specific adducts of the ATE-AO type. The above results were supported by differential UV–Vis spectroscopy measurements, which revealed the formation of new bands around 370 nm, attributed to the formation of the adducts mentioned above. The presented work describes the first characteristics of chemiluminogenic acridinium thioesters and draws perspectives on their practical uses in the analytics of biological substances. These findings also contribute to our understanding of the reactivity of ATEs in solutions accompanied by light emission. •Synthesis and spectroscopic features of new chemiluminogenic salts, acridinium thioesters (ATEs), are reported.•The efficiency and kinetics of chemiluminescence of ATEs were evaluated in various liquid environments containing H2O2/KOH.•Usefulness of the new family of compounds was demonstrated in luminescence analysis of biological antioxidants (AOs).•DFT studies indicated the mechanism of CL with the formation of ATE-AO adducts.•Differential UV–Vis spectroscopy proved the formation of ATE-AO adducts.