Silver nanoparticles (AgNPs) are some of the most attractive nanomaterials for commercial applications, but they can also be a very challenging issue from the point of view of cytotoxicity and phytotoxicity as agents that damage genetic information within a cell causing mutations. AgNPs can be easily biosynthesized mainly by fungi which release enzymes in response to stress conditions, reducing silver ions to nanoscale size. Nowadays, mycogenic AgNPs are becoming increasingly relevant for several industrial and environmental applications, therefore, we urgently need to understand in detail how these AgNPs act and their potential effects on the environment to take measures to prevent the occurrence of hazards. The present work had the goal of evaluating the effect of spherical AgNPs synthesized by Penicillium citrinum IBCLP11 (AgNPsIBCLP11), average diameter of 57.5 ± 4 nm, against pathogenic bacteria Pseudomonas aeruginosa IPT322, Staphylococcus aureus IPT246 and Klebsiella pneumoniae IPT412 and their potential toxicity to the freshwater organisms Chlorella vulgaris, Palaemon pandaliformis and Danio rerio. AgNPsIBCLP11 presented the minimum inhibitory concentration for IPT322 strain of 15 µg•mL-1, while for IPT246 and IPT412, it ranged from 20-25 µg•mL-1. For all freshwater organisms studied, the AgNPs toxicity was lower than their metal precursor. Chlorella vulgaris growth and survival rates after 72 h of exposure to AgNPsIBCLP11, were not affected at concentrations ranging from 10 to 100 μg•L-1. AgNPsIBCLP11 and silver nitrate (AgNO3) caused no acute toxicity on adult zebrafish, although they did induce several physiological changes. In view of the results obtained, the AgNP showed promising potential for their use in freshwater systems decontamination, especially against pathogenic microorganisms, making it possible to reduce deleterious impacts on the ecosystem and enabling the conservation and management of this natural resource.