The increasing production of eco-friendly nanoparticles like biosynthesized nanoparticles (BNPs) calls for study on their environmental and biological safety. Herein, the impact of natural organic matter on the toxicity of BNPs was studied. Using leaf extract of herbal plant Allium fistulosum, the Allium fistulosum-silver nanoparticles (AF-AgNPs) were synthesized with the yield of around 100% and used to explore the impacts of natural organic matter (Suwannee river humic acid) on their toxicity to green microalgae Chlorella vulgaris. The results showed that the as-prepared AF-AgNPs could decrease the end-points of biomass and chlorophyll a content of C. vulgaris in a dose-dependent manner. In addition, AF-AgNPs enhanced algal aggregation and decreased size of cells, especially at higher concentrations. However, organic matter showed an ameliorative effect on the toxicity of AF-AgNPs, and significant enhancement of biomass and chlorophyll a content (p < 0.05) were observed in media treated with higher contents of AF-AgNPs. Organic matter could also prevent more cellular aggregation and size reduction of C. vulgaris. Our results are helpful for understanding the effects of organic matter on the toxicity of BNPs to aquatic organisms.