In this study, the selection of bacteria on the basis of their migration via fungal hyphae in soil was investigated in microcosm experiments containing Lyophyllum sp. strain Karsten (DSM2979). One week following inoculation with a bacterial community obtained from soil, selection of a few specific bacterial types was noticed at 30 mm in the growth direction of Lyophyllum sp. strain Karsten in sterile soil. Cultivation-based analyses showed that the migration-proficient types encompassed 10 bacterial groups, as evidenced by (GTG)(5) genomic fingerprinting as well as 16S rRNA gene sequencing. These were (>97% similarity) Burkholderia terrae BS001, Burkholderia sordidicola BS026, Burkholderia sediminicola BS010, and Burkholderia phenazinium BS028; Dyella japonica BS013, BS018, and BS021; "Sphingoterrabacterium pocheensis" BS024; Sphingobacterium daejeonense BS025; and Ralstonia basilensis BS017. Migration as single species was subsequently found for B. terrae BS001, D. japonica BS018 and BS021, and R. basilensis BS017. Typically, migration occurred only when these organisms were introduced at the fungal growth front and only in the direction of hyphal growth. Migration proficiency showed a one-sided correlation with the presence of the hrcR gene, used as a marker for the type III secretion system (TTSS), as all single-strain migrators were equipped with this system and most non-single-strain migrators were not. The presence of the TTSS stood in contrast to the low prevalence of TTSSs within the bacterial community used as an inoculum (<3%). Microscopic examination of B. terrae BS001 in contact with Lyophyllum sp. strain Karsten hyphae revealed the development of a biofilm surrounding the hyphae. Migration-proficient bacteria interacting with Lyophyllum sp. strain Karsten may show complex behavior (biofilm formation) at the fungal tip, leading to their translocation and growth in novel microhabitats in soil.