Endophyte fungi (EF) are considered a new and valuable reservoir of bioactive molecules of biotechnological interest for pharmacy, agricultural and forestry industries. In this study, thirty EFs, isolated from three Chilean Nothofagus species (N. alpina, N. dombeyi, N. oblicua) were identified and cultured in submerged liquid fermentations aimed at searching for natural active substances. The extracts obtained were evaluated against pathogenic bacteria and fungi. Sixteen extracts (53.3%) presented antibacterial and fourteen (46.6%) presented antifungal activities in different intensities. Extracts from isolates Coryneum sp.-72 and P. cinnamomea-78 exhibited the highest antimicrobial activity. Using bioautography, the compounds responsible for the antimicrobial activity exhibited by Coryneum sp.-72 and P. cinnamomea-78 were detected and characterized. Coryneum sp.-72 showed bactericidal properties at 200 μg/mL and bacteriostatic effects at 50 μg/mL against B. cereus, B. subtilis, L. monocytogenes and S. aureus. MIC values indicated that P. cinnamomea-78 exhibited a strong fungistatic and fungicidal effect against B. cinerea and C. gloesporioides at 10–50 μg/mL. Isolates were grouped in the following order: Botryosphaeriales, Diaporthales, Eurotiales, Helotiales, Hypocreales, Pleosporales, Magnaporthales, Sordariales and Polyporales. EF isolated, identified and evaluated constitute the first report for Chilean Nothofagus genus.