Microbial resistance to antibiotics is one of the key challenges that lead to the search for alternate antimicrobial treatment approaches. Silver nanoparticles (AgNPs) are well known for their antimicrobial effects against a wide variety of drug-resistant microorganisms. AgNPs can be synthesized using microbial hosts, using a green and economical synthesis route, which produces extremely stable and highly active nanoparticles. Such green AgNPs are coated with a biological coating often referred to as a corona, originating from the production microorganism. In this study, we asked whether the composition of the biological corona might influence the antimicrobial activity of green AgNPs. To investigate this, we produced AgNPs in Pseudomonas putida KT2440 and Escherichia coli K12 MG1655, and tested them against pathogen species from the corresponding genera. AgNPs exhibited a size range of 15–40 nm for P. putida and 30–70 nm for E. coli, and both types of nanoparticles were surrounded by a thick biological corona layer, providing extreme stability. The nanoparticles remained stable over long periods and exhibited negative zeta potential values. P-AgNPs (obtained from P. putida) were tested against pathogenic Pseudomonas aeruginosa PAO1, and E-AgNPs (obtained from E. coli) were tested against pathogenic Escherichia coli UTI 89. Antimicrobial studies were conducted by Minimum bactericidal concentration (MBC), live/dead staining and SEM analysis. MBC of P-AgNPs against P. aeruginosa was 1 μg/mL, and MBC of E-AgNPs against E. coli UTI 89 was 8 μg/mL. In both cases, the MBC values were superior to those of green AgNPs produced in organisms unrelated to the target pathogens, available in the literature. Our results suggest that NPs produced in microorganisms closely related to the target pathogen may be more effective, indicating that the composition of the biological corona may play a crucial role in the antimicrobial mechanism of AgNPs.