Antimicrobial resistance is considered a problem for public health globally. New forms of resistance mechanisms are developing and spreading daily around the world. For that reason, a potential method for overcoming the antimicrobial resistance of several pathogens that cause deadly infections is the use of silver nanoparticles. In the present study, silver nanoparticles (AgNPs) and AgNPs conjugated with lincomycin were synthesized and characterized. The antimicrobial activity of AgNPs alone and after conjugating with lincomycin was also evaluated. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were employed to determine the morphological properties of AgNPs and AgNPs-lincomycin, which showed the average mean size was ±26.73 nm for AgNPs and ±28.31 nm for AgNPs-lincomycin with a spherical shape. In addition, the dynamic light scattering (DLS) and zeta potential were assessed. The results showed an improvement in the antimicrobial activity of lincomycin after conjugation with AgNPs, as the inhibition zone diameter reached 45.66±1.52 mm for Staphylococcus warneri, 17±2 mm for Serratia marcescens, and 22.33±1.52 mm for Candida guilliermondii. It also showed a synergistic effect of AgNPs-lincomycin against the biofilm formation of Candida guilliermondii, whereas Serratia marcescens was slightly affected. The MIC of AgNPs was 25 μg/ml for Staphylococcus warneri and Candida guilliermondii, whereas 100 μg/ml for Serratia marcescens, when the MIC of AgNPs-lincomycin was 12.5 μg/ml for Staphylococcus warneri and Candida guilliermondii, and 100 μg/ml for Serratia marcescens. In the experiment of the effect of AgNPs on gene expression of the antibiotic resistance genes, including the blaZ gene in Staphylococcus warneri, the aac(6´)-Ib-cr gene in Serratia marcescens, and the CDR1 gene in Candida guilliermondii, the results showed that there was a decrease in gene expression after being treated with AgNPs in each gene.