Here we tailored a methodology, including green synthesis of silver nanoparticles (AgNPs) in aqueous solution using Fluconazole (Fluc.), a broad-spectrum antifungal agent under the influence of gamma rays. AgNPs were characterized by UV–Vis., FTIR, XRD, DLS, and TEM image. Antimicrobial activities of AgNPs, Fluc., and Ag+ were investigated against multidrug-resistant (MDR) bacteria and unicellular fungi. From our results, AgNPs production was found to be dependent on the concentration of Ag+, Fluc. and gamma doses. DLS with TEM image explained the size and shape of AgNPs and were found to be spherical with diameter of 11.65 nm. FTIR analysis indicates that, the hydroxyl, nitrogen and fluoride moiety in Fluc. were responsible for the reduction and binding process. AgNPs possesses antimicrobial activity against all tested microbes more than Ag+. It produced high efficacy against Acinetobacter baumannii (20.0 mm ZOI). AgNPs are synergistically active towards Candida albicans (17.0 mm ZOI). Investigated action mechanisms for AgNPs activity had been discussed. Thereby, owing to its unique features as cost-effective with continued-term stabilization, it can discover potential targets in biomedical applications and infectious diseases control.