Mushroom extracts have shown potential as a source of new antimicrobial agents. This study investigates the chemical profile of an aqueous ammonia extract obtained from the carpophores of Ganoderma lucidum, which grows on Quercus ilex trees, and explores its valorization as a biorational. The major chemical constituents of the extract, identified through gas chromatography–mass spectrometry, include acetamide, oleic acid, 1,2,3,4-butanetetrol, monomethyl azelate, undecane, and palmitic acid. The anti-oomycete and antifungal activity of G. lucidum extract was evaluated against Phytophthora cinnamomi, the primary threat to Quercus spp. in the dehesa biome, as well as three Botryosphaeriaceae fungi. In vitro tests revealed minimum inhibitory concentration (MIC) values of 187.5 μg·mL−1 against P. cinnamomi and 187.5–1000 μg·mL−1 against the fungi. Furthermore, conjugation of the G. lucidum extract with chitosan oligomers (COS) synergistically enhanced its antimicrobial activity, resulting in MIC values of 78.12 and 375–500 μg·mL−1 against P. cinnamomi and the fungi, respectively. These MIC values are among the highest reported to date for natural products against these phytopathogens. Subsequent ex situ testing of the COS-G. lucidum conjugate complex on artificially inoculated Q. ilex excised stems resulted in high protection against P. cinnamomi at a dose of 782 µg·mL−1. These findings support the potential utilization of this resource from the dehesa ecosystem to protect the holm oak, aligning with sustainable and circular economy approaches.
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