||Abstract The quality of torrefied grass fibers (TGF) as a new potting soil ingredient was tested in a greenhouse experiment. TGF was colonized with previously selected microorganisms. Four colonization treatments were compared: (1) no inoculants, (2) the fungus Coniochaeta ligniaria F/TGF15 alone, (3) the fungus followed by inoculation with two selected bacteria, and (4) the fungus with seven selected bacteria. Cultivation-based and DNA-based methods, i.e., PCR-DGGE and BOX-PCR, were applied to assess the bacterial and fungal communities established in the TGF. Although colonization was not performed under sterile conditions, all inoculated strains were recovered from TGF up to 26 days incubation. Stable fungal and bacterial populations of 108 and 109¿CFU/g TGF, respectively, were reached. As a side effect of the torrefaction process that aimed at the chemical stabilization of grass fibers, potentially phytotoxic compounds were generated. These phytotoxic compounds were cold-extracted from the fibers and analyzed by gas chromatography mass spectrometry. Four of 15 target compounds that had previously been found in the extract of TGF were encountered, namely phenol, 2-methoxyphenol, benzopyran-2-one, and tetrahydro-5,6,7,7a-benzofuranone. The concentration of these compounds decreased significantly during incubation. The colonized TGF was mixed with peat (P) in a range of 100%:0%, 50%:50%, 20%:80%, and 0%:100% TGF/P (w/w), respectively, to assess suitability for plant growth. Germination of tomato seeds was assessed three times, i.e., with inoculated TGF that had been incubated for 12, 21, and 26 days. In these tests, 90–100% of the seeds germinated in 50%:50% and 20%:80% TGF/P, whereas on average only 50% of the seeds germinated in pure TGF. Germination was not improved by the microbial inoculants. However, plant fresh weight as well as leaf area of 28-day-old tomato plants were significantly increased in all treatments where C. ligniaria F/TGF15 was inoculated compared to the control treatment without microbial inoculants. Colonization with C. ligniaria also protected the substrate from uncontrolled colonization by other fungi. The excellent colonization of TGF by the selected plant-health promoting bacteria in combination with the fungus C. ligniaria offers the possibility to create disease suppressive substrate, meanwhile replacing 20% to 50% of peat in potting soil by TGF.