Aims: To evaluate the inactivation of Bacillus anthracis Delta Sterne and Ames spores using electrochemically generated liquid-phase chlorine dioxide (eClO(2)) and compare two sporulation and decontamination methods with regard to cost, safety and technical constraints. Methods and Results: Spores were prepared via agar and broth methods and subsequently inoculated and dried onto clean, autoclave-sterilized glass coupons. Bacillus anthracis spore inactivation efficacy was evaluated using the modified three-step method (AOAC 2008.05) and a single-tube extraction method. Spores (7.0 +/- 0.5 logs) were inactivated within 1 min at room temperature using freshly prepared eClO(2). Bacillus anthracis Delta Sterne spores decreased in size after eClO(2) treatment as measured using a Beckman Coulter Multisizer. Conclusions: eClO(2) saturation of a hard surface was an effective B. anthracis sporicide. Broth sporulation and the single-tube extraction method required less time and fewer steps, yielded a higher percentage of phase-bright spores and showed higher spore recovery efficiency compared with AOAC 2008.05, making it more amenable to biosafety level 3 (BSL3) testing of virulent spores. Significance and Impact of the Study: Two test methods demonstrated the sporicidal efficacy of eClO(2). A new single-tube extraction test protocol for decontaminants was introduced.