**Background:** The rise of antibiotic-resistant infections, oxidative stress-related chronic diseases, and neurodegenerative conditions like Alzheimer's disease has driven the search for novel bioactive compounds from underexplored sources. Marine microorganisms, particularly fungi from extreme environments, are promising producers of unique secondary metabolites. Penicillium chrysogenum is well-known for penicillin production, but its potential for other bioactivities—especially from halotolerant marine strains—remains underexplored. This study aimed to evaluate the antimicrobial, antioxidant, and acetylcholinesterase (AChE) inhibitory activities of the non-polar ethyl acetate extract of Penicillium chrysogenum MZ945518 isolated from the Mediterranean Sea, and to characterize its metabolite profile and conduct in silico analyses.
**Methods:** The fungus was isolated from the Mediterranean coast of Alexandria, Egypt, and identified molecularly. Halotolerance was assessed by measuring colony diameter on PDA supplemented with 0–30% NaCl. Mycelia were extracted with ethyl acetate, and the extract was tested for: (1) antibacterial activity against six bacterial strains (Pseudomonas aeruginosa ATCC 7853, Proteus mirabilis ATCC 29906, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619, Micrococcus luteus ATCC 9341) and anticandidal activity against Candida albicans ATCC 20231 using agar diffusion; (2) antifungal activity against Rhizoctonia solani, Fusarium oxysporum, and Fusarium solani by measuring percent inhibition of mycelial growth (PIMG); (3) antioxidant activity via DPPH free radical scavenging, ferric reducing antioxidant power (FRAP), and metal ion chelating assays; and (4) AChE inhibitory activity using Ellman's method with donepezil as standard. Total phenolics and flavonoids were quantified colorimetrically. GC/MS identified 20 metabolites. Molecular docking was performed against DNA Gyrase (6M1J), glutathione S-transferase (13GS), and AChE (1ACJ). ADMET profiling used SwissADME.
**Key Results:** The fungus was halotolerant (Ti = 1.3 at 5% NaCl). The extract showed strongest antibacterial activity against Proteus mirabilis ATCC 29906 (inhibition zone 20 mm vs. 12 mm for gentamycin) and Micrococcus luteus ATCC 9341 (12 mm vs. 10 mm). Antifungal activity was highest against Fusarium solani (PIMG = 77.5 ± 0.3%), followed by Rhizoctonia solani (52 ± 0.0%) and Fusarium oxysporum (40 ± 0.5%). DPPH scavenging IC50 was 542.5 ± 69.1 µg/mL. FRAP showed Fe³⁺ reduction, and metal chelating activity was 12.7 ± 0.9 µM EDTA eq/mg extract. AChE inhibition was 63% with IC50 = 60.87 ± 3.8 µg/mL. Total phenolics were 373.5 mg GAE/g and flavonoids 133.4 mg QE/g. GC/MS identified 20 metabolites; major ones were (Z)-18-octadec-9-enolide (36.28%), 1,2-Benzenedicarboxylic acid (26.73%), n-hexadecanoic acid (7.8%), 2,3-dihydroxypropyl acetate (5.3%), 9,12-octadecadienoic acid methyl ester (4.8%), and butyl 9,12,15-octadecatrienoate (3.2%). Docking showed compounds 2, 6, and 8 had highest binding energies against DNA Gyrase (−7.78, −7.33, −7.76 kcal/mol). Against AChE, 2,3-dihydroxypropyl acetate had the highest binding efficacy (ΔG = −8.72 kcal/mol). ADMET profiling indicated most compounds had favorable pharmacokinetic properties and complied with Lipinski's rule of five.
**Clinical Implications:** The study demonstrates that halotolerant Penicillium chrysogenum MZ945518 produces a diverse array of bioactive metabolites with potential therapeutic applications. The extract's strong activity against Proteus mirabilis (a urinary tract pathogen) and Micrococcus luteus suggests potential for developing new antibacterial agents, particularly against strains where conventional antibiotics like gentamycin are less effective. The antifungal activity against Fusarium species—important plant and opportunistic human pathogens—highlights biocontrol potential. The AChE inhibitory activity (63% inhibition, IC50 60.87 µg/mL) positions this extract as a candidate for further investigation in Alzheimer's disease research, as AChE inhibition is a validated therapeutic strategy. The antioxidant activity, though moderate (DPPH IC50 542.5 µg/mL), may contribute to neuroprotective effects. The in silico docking and ADMET results provide a molecular basis for these activities and suggest favorable drug-like properties for several metabolites. However, these are in vitro and in silico findings; in vivo validation, toxicity studies, and isolation of individual active compounds are needed before clinical translation.