ABSTRACT The chemical and physical techniques applied for nanoparticles synthesis are mostly high cost and may have harmful effects. So, there is an indigence to synthesize a low cost, high yield, most effective and most stable nanoparticles. The antimicrobial activity of mycosynthesized titanium oxide (TiO2NPs) and silver (AgNPs) nanoparticles from mycelial extract of Penicillium chrysogenum AUMC 6092 was the main target of this study. The antimicrobial activity was assayed against 5 pathogenic Gram- positive (Staphylococcus aureus and Streptococcus sp.) and Gram- negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) bacteria. In addition, 12 human pathogenic fungal strains including some multidrug resistant yeast strains and dermatophytic fungal strains (Epidermophyton floccosum, Microsporum canis, Trichophyton mentagrophytes and T. rubrum) were tested. TiO2NPs and AgNPs synthesized from culture filtrate of Penicillium chrysogenum AUMC 6092 are uniform, spherical and small. The results showed that, generally TiO2NPs and AgNPs had high inhibitory activity against most tested microorganisms, especially when mixed with Tioconazole. TiO2NP2 had the highest inhibitory effect against C. glabrata AUMC 13382, followed by Escherichia coli, Staphylococcus aureus and C. tropicalis AUMC 13378. On the other hand, Staphylococcus aureus and C. glabrata AUMC 13382 were the most sensitive microbial strains towards AgNPs.