•XRCT and endoscopy as new approach to investigate biopore characteristics.•Highly advantageous characterization of structural elements (blockages, linings).•Observation of dynamic development of pore network after introduction of earthworms in microcosms.•Rate of colonization of existing biopores by earthworms is higher than rate of newly formed pores.•Favorable effects on the nutrient contents of the biopore walls as result of fresh earthworm cast. Bioporosity in subsoils is strongly influenced by either deep rooting taproots or earthworms, which can affect important properties for plant growth provided by soils. The open question of this study was, in how far the combination of X-ray computed tomography (XRCT) and endoscopy may support the analysis of soil bioporosity and the effects induced by earthworm activity. The hypothesis were that earthworms can re-open, close and re-connect pores as well as change the biopore wall properties (by leaving coatings at the wall) and that with a combination of XRCT and endoscopy much more detailed information about those changes can be observed. Soil monoliths were collected from a Haplic Luvisol (developed from loess) at the experimental station Klein Altendorf (University of Bonn, Germany). The microcosms were then prepared under laboratory conditions in terms of temperature, soil temperature gradient, relative humidity, illumination, watering and fertilizer. The microcosms were examined before cultivation and incubation, and re-examined after one-month earthworm incubation (Lumbricus terrestris L.), three months wheat growth period and shoot harvesting. XRCT and endoscopy were used to qualitatively and quantitatively analyze the soil microcosms. After the incubation with earthworms, physical properties like porosity and accessible surface area of biopores have changed with respect to the original state. Coatings at the biopore walls resulted in smaller pore diameters of large biopores with a diameter >0.5cm, while some of them have previously been pores with diameters <0.5cm. Contrary to this, biopores with a diameter <0.5cm were increased after earthworms, i.e. widened due earthworm passage. It was found that earthworms may have disconnected lateral pores that reach into the bulk soil from the vertical biopores by pore wall coatings. After incubation, biopores lined with fresh earthworm coatings in the monolith increased from 30% to 80%, which may indicate that earthworms potentially have a considerable influence on biopore properties and therefore the physical, chemical and microbiological environment which roots are exposed to in biopores.