Bacillus anthracis is one of the most dangerous and pathogenic bacterial species and its intrusion in aquatic environments is a serious threat to public health. The aim of the present study was to investigate inactivation rates of B. anthracis in water by means of photocatalytic (UVA/TiO), photolytic (UVC) and sonochemical treatment. The effect of various operating conditions such as bacterial concentration, TiO loading, UV irradiation source, ultrasound power and treatment time was examined. The reference strain of B. anthracis proved to be highly resistant during photocatalytic and sonochemical treatment of aquatic samples, even in the presence of hydrogen peroxide solution, which is considered among the chemical disinfectants recommended for B. anthracis removal from aqueous suspensions. UVC irradiation was far more effective, as it achieved total inactivation in short treatment time (10 min) and at high initial concentrations (10 CFU mL). The effectiveness of UVC irradiation is also reinforced by the fact that no photoreactivation occurred even after 72 h of exposure under sunlight after the end of the treatment. Furthermore, the virulence of residual cells was investigated, targeting two genes carried in the plasmids pXO1 and pXO2, respectively, which are required for a fully virulent type. Interestingly, the plasmid pXO2 seems to be lost from the host after photocatalytic and photolytic disinfection, resulting in apathogenic residual strains contained in the treated sample. Overall, our results highlight the importance of B. anthracis efficient inactivation in water, as it shows considerable resistance towards effective and reliable disinfection techniques.