Cellular interactions and catabolic activities of mycorrhizal root associated non-sporulating bacteria were investigated in a simplified phytoremediation simulation involving a woody plant species. Mycorrhizal Scots pine (Pinus sylvestris) seedlings pre-colonised by Suillus bovinus or Paxillus involutus were grown in forest humus containing microcosms amended with petroleum hydrocarbon (PHC) contaminated soil. Fungal hyphae of both species, emanating from mycorrhizal roots, colonised the PHC contaminated soil over a 16-week period and dense long-lived patches of S. bovinus hyphae formed on the PHC contaminated soil. Transmission electron microscopy revealed a microbial biofilm at the PHC soil-fungal interface composed of differentiated pseudoparenchymous patch hyphae supporting a morphologically diverse bacterial population. Certain non-sporulating bacterial isolates closely associated with the S. bovinus patch hyphae or P. involutus‘web’ hyphae from the PHC soil harboured similar sized mega-plasmids (approx. 150 kb). Isolates of Pseudomonas fluorescens from the ‘patch’ mycorrhizospheres represented different biovars, displayed similar REP-PCR genomic fingerprints, grew on e.g. m-toluate and m-xylene as sole carbon sources, cleaved catechol, and harboured plasmid-borne catabolic marker genes, xylE and xylMA, involved in degradation of mono-aromatics. The plasmids were transmissible in vitro, and Pseudomonas putida transconjugants retained a similar catabolic profile. The identification of microbial biofilms containing catabolic bacteria in the external mycorrhizosphere is discussed in relation to both phytoremediation mechanisms and normal efficient nutrient mobilisation from highly lignin-rich forest soils.