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<i>Pinus sylvestris</i> bark represents a rich source of active compounds with antifungal, antibacterial, and antioxidant properties. The current study aimed to evaluate the antifungal potential of <i>P. sylvestris</i> bark against <i>Botryosphaeria dothidea</i>, <i>Dothiorella sarmentorum</i>, and <i>Neofusicoccum parvum</i> (Botryosphaeriaceae) through its chemical (water extracts) and biological (<i>Trichoderma</i> spp. isolated from the bark) components. The water bark extracts were prepared at two temperatures (80 and 120 °C) and pH regimes (7 and 9). The presence of bark extracts (30%) caused inhibition of mycelial growth of <i>B. dothidea</i> and <i>D. sarmentorum</i> for 39 to 44% and 53 to 60%, respectively. Moreover, we studied the antagonistic effect of three <i>Trichoderma</i> isolates originating from the pine bark. <i>Trichoderma</i> spp. reduced growth of <i>B. dothidea</i> by 67%–85%, <i>D. sarmentorum</i> by 63%–75% and <i>N. parvum</i> by 55%–62%. Microscopic examination confirmed typical mycoparasitism manifestations (coiling, parallel growth, hook-like structures). The isolates produced cellulase, β-glucosidase and N-acetyl-β-glucosaminidase. The volatile blend detected the emission of several volatile compounds with antimicrobial activity, including nonanoic acid, cubenene, cis-α-bergamotene, hexanedioic acid, and verticillol. The present study confirmed in vitro potential of <i>P. sylvestris</i> bark extracts and <i>Trichoderma</i> spp. against the Botryosphaeriaceae. The study is an important step towards the use of environmentally friendly methods of Botryosphaeriaceae disease control.