X-ray Microfocused Computed Tomography (X-ray μ-CT) allows a non-destructive and three-dimensional observation of microbial habitats (i.e. pore space) in soil. A major premise for microbiological studies integrating X-ray μ-CT is that soil microorganisms are not affected by irradiation dose in terms of physiology and composition. However, the compatibility of X-ray μ-CT and soil biological experiments has been evaluated controversially.We performed an incubation experiment with packed microcosms to assess the effect of X-ray μ-CT on native microbial populations with emphasis on soil archaea and bacteria. Before (14 days) and after (1 and 14 days) scanning we analyzed (i) respiration, (ii) enzyme activity, (iii) microbial biomass, (iv) abundance and (v) community structure in scanned and control treatments.None of the microbial parameters exhibited significant differences among scanned and unscanned soil samples at all sampling times with the exception of lower archaeal cell numbers subsequent to X-ray μ-CT. Incubation time was the main factor that induced a significant alteration of microbial soil populations while irradiation had no or only very little effect thereupon. Taken together, three-dimensional in situ data obtained via X-ray μ-CT may well be combined with microbiological analyses in soil.