Mercury (Hg) is a heavy metal that is commonly found in waters and sediments. Mercury and its derivatives are highly toxic). Their presence in the aquatic environment can be very detrimental. Aspergillus fungi are known to reduce Hg levels in pollutants. Our research aim was to evaluate the toxic effect of Hg after being reduced by local isolates of Penicillium sp. strain A4. A toxicity test was conducted on juvenile Caroline platyfish (Xiphophorus maculatus). The research was conducted by growing fungal isolates on potato dextrose broth (PDB) medium with the addition of Hg at 7 days of incubation. The Hg concentration was measured before and after the incubation. The acute toxicity test was conducted with two serial concentrations: 50% and 100% of PDB-Hg media from fungal removal, as well as one control. The acute toxicity test was carried out for 48 hours and the number of deaths of Xiphophorus maculatus was counted. The results showed a decrease of mercury concentration from 3.14 ppm to 0.797 ppm in PDB-Hg media on the activity of isolates of Penicillium sp. strain A4 resulting in 74.6% mercury removal. Acute toxicity test results showed that concentrations of 50% and 100% of PDB-Hg media caused 100% death of the fish. The results of this study can be used as initial data to determine the quality criteria for waste containing mercury that can be discharged into river bodies and strategies for using fungi applications using local isolates of Penicillium sp. strain A4, which has the potential for wastewater treatment before being discharged into the waters to reduce its toxic effects.