Biodegradation of organic compounds in soil is a key process that has major implications for chemical fertility and plant nutrition, physical properties, carbon storage, control of pathogens and also dynamics of pollutants. The fate of organic compounds in soil is controlled by processes occurring at the micro-scale. Measurements and modeling performed on a macroscopic scale do not take into account the microscopic specificities of the soil and in particular the important heterogeneity at this scale. If microorganisms and substrates are not co-localized, transfer processes controlling the accessibility of the microbial substrate will be a key factor. Active motility of bacteria is generally reduced in soil. Therefore, bacterial degradation is strongly regulated by diffusive/convective transport of substrates to these microorganisms. The objectives of this work were to better understand how transport conditions drive spatial distribution of 2,4-D and its bacterial degraders at a millimetric scale. We built soil columns (diameter 5 cm, height 3 cm) combining natural and sterilized soil aggregates in which we localized soil microorganisms and the pesticide 2,4-D (2,4-Dichlorophenoxyacetic acid), with the following spatial distributions: (i) homogeneous distribution of microorganisms and 2,4-D in all the soil cores, (ii) co-localized distribution of microorganisms and 2,4-D in a mm3 spot and iii) a disjoint localization of microorganisms and 2,4-D in 2 mm3 soil spots. Controls with only 2,4-D or only microorganisms were also carried out. Two sets of experiments were performed: one with 14C-2,4-D to study the fate of 2,4-D and one with 12C-2,4-D to follow the growth of degraders, total bacteria and fungi. Microcosms were incubated at 20°C and at a matrix potential of -320 cm (pF 2.5). Three irrigation events corresponding to 2 pore volumes were carried out every 3 days after starting the incubation. At the core scale we followed total and 2,4-D mineralization (CO2 and 14CO2) with time and measured 14C in leachates. After 24 days of incubation, soil cores were cut out in slices of 6 mm thickness. Each slice was then further separated into soil cubes (6 mm edge length). The individual soil cubes were analyzed for extractable and non-extractable 14C-residues and for microorganisms (qPCR of tfdA genes, 16S and 18S rRNA genes). Knowing the initial position of soil cubes allowed us to establish 3D maps of 14C residues and microorganisms in soil. Results showed that whatever the spatial distribution of 2,4-D and microorganisms, the loss of 14C in leachates represented more than 50% of added 14C and was not significantly different between treatments. The 2,4-D mineralization rates were faster when 2,4-D and microorganisms were homogeneously distributed in the entire soil core. When substrate and microorganisms were co-localized at the top of the core, 2,4-D mineralization rates were slower but the final mineralization was close to the homogenous distribution after 24 days (28.7 and 32.7% of added 14C respectively). When 2,4-D was localized at the top and microorganisms at the bottom of the soil core, 2,4-D mineralization represented only 11.4% of added 14C at the end of the incubation. The disappearance of 14C in the extractable fraction and the production of non-extractable 14C residues were stronger when mineralization increased. For the homogeneous distribution of 2,4-D and microorganisms, we detected degraders, bacteria and fungi in the entire core. When 2,4-D and microorganisms were not co-localized, microorganisms were concentrated at the bottom of the soil cores while 2,4-D was distributed by diffusion and convection everywhere in the core. When 2,4-D and microorganisms were co-localized at the top of the soil core, 2,4-D spread in the entire soil core, whereas microorganisms grew or were transferred mainly vertically in the central part of the core. Non-extractable residues were mainly produced in zones colonised by microorganisms. Bacterial degraders of 2,4-D played a predominant role in the formation and localization of biogenic non-extractable residues in soil. These results emphasize the importance of considering microscale processes to better understand the fate of organic substrates in soil.