Antibiotic resistance has substantially risen worldwide. Considering this worrying condition, many studies on biogenic nanoparticles and their antimicrobial and anticancer properties have been conducted. There are numerous ways to improve stability and dispersion through conjugate peroration with bioactive natural ingredients such as chitosan. Here, a fungal isolate was isolated and identified as Talaromyces sp. 7S1 by 18SrRNA with accession no. OP295112. This strain was used to prepare biogenic silver nanoparticles (AgNPs) and chitosan–AgNPs conjugate (Ch-AgNPs). A variety of imaging and analytical methods including UV–vis, XRD, FTIR-spectroscopy, TEM, and SEM were utilized to characterize biogenic nanoparticles and conjugates. The biosynthesized AgNPs along with the prepared conjugate were evaluated for their antimicrobial effects on Gram-positive (Staphylococcus aureus (RCMB 010028) and Bacillus subtilis (RCMB 010067)) and Gram-negative (Escherichia coli )RCMB 010052) and Salmonella typhi)RCMB 010052)) pathogens. Ch–AgNPs showed good antimicrobial activity compared to control. Results revealed that, the crude extract showed antibacterial activity against E. coli and S. aureus while a substantial effect was observed with AgNPs. AgNPs displayed potent antibacterial activity against E. coli, S. aureus, and B. subtilis, while it showed moderate activity against S. typhi. Furthermore, Ch-AgNPs exhibited a pronounced antibacterial activity against E. coli, and S. aureus and low antibacterial activity against S. typhi. On the other hand, the obtained AgNPs and Ch-AgNPs exhibited a pronounced antibiofilm activity against all tested strains. Additionally, the antioxidant and anticancer activities of AgNPs and Ch-AgNPs were also evaluated. Collectively, AgNPs and Ch-AgNPs exhibit good antimicrobial activity, nevertheless, Ch-AgNPs conjugate reduced the toxicity of AgNPs which may represent a safer tool to inhibit bacterial pathogens.