This work was focused on the characterization of novel isolate of Trichoderma atroviride O1, found in the forest around the village of Zázrivá (the Northern Slovakia, region Orava). The isolate was identified by sequencing its internal transcribed spacer (ITS) region of rDNA. T. atroviride O1 stimulated the development of lateral roots of model plant Lepidium sativum. Simultaneously, the isolate has proved its high mycoparasitic potential as it displayed the ability to attack colonies of phytopathogenic fungi (Alternaria alternata, Fusarium culmorum, Botrytis cinerea). This isolate produced secondary metabolites, which were isolated and tested for the antimicrobial activity against gram-positive bacteria Staphylococcus epidermidis and Staphylococcus aureus. The growth of these bacteria was suppressed to 10 % and 40 %, respectively. The suppression of the growth of two Candida species was also strong (10 % growth). However, growth parameters of three phytopathogenic fungi (Alternaria alternata, Botrytis cinerea and Fusarium culmorum) were less affected (75 % growth in comparison with the control). Attempts were made to characterize secondary metabolites isolated from T. atroviride O1. Known peptaibols, 20—21 amino acid long, but also shorter peptides, were detected by MALDI-TOF mass spectrometry. Thus, this study demonstrates the plant growth promotion, strong mycoparasitic potential and antimicrobial activity of the isolate T. atroviride O1, which could be in part ascribed to the production of secondary metabolites. This isolate does have a potential in the biocontrol in eco-farming. Further study, particularly, the identification of produced secondary metabolites, is needed.