Aims: To compare the killing efficacy and the effects exerted by microwaves and conventional heating on structural and molecular components of Bacillus subtilis spores. Methods and Results: A microwave waveguide applicator was developed to generate a uniform and measurable distribution of the microwave electric-field amplitude. The applicator enabled the killing efficacy exerted by microwaves on B. subtilis spores to be evaluated in comparison with conventional heating at the same temperature value. The two treatments produced a similar kinetics of spore survival, while remarkably different effects on spore structures were seen. The cortex layer of the spores subjected to conductive heating was 10 times wider than that of the untreated spores; in contrast, the cortex of irradiated spores did not change. In addition, the heated spores were found to release appreciable amounts of dipicolinic acid (DPA) upon treatment, while extracellular DPA was completely undetectable in supernatants of the irradiated spores. These observations suggest that microwave radiation may promote the formation of stable complexes between DPA and other spore components (i.e. calcium ions); thus, making any release of DPA from irradiated spores undetectable. Indeed, while a decrease in measurable DPA concentrations was not produced by microwave radiation on pure DPA solutions, a significant lowering in DPA concentration was detected when this molecule was exposed to microwaves in the presence of either calcium ions or spore suspensions. Conclusions: Microwaves are as effective as conductive heating in killing B. subtilis spores, but the microwave E-field induces changes in the structural and/or molecular components of spores that differ from those attributable only to heat. Significance and Impact of the Study: This study provides information on the effect of microwaves on B. subtilis spore components.