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This study demonstrated the effective reduction of graphene oxide (GO) by employing thiourea as a reducing and stabilizing agent. Two fungi (<i>Aspergillus flavus</i> and <i>Aspergillus fumigatus</i>) were used for anti-fungal assay. Cell viability, cell cycle analysis, DNA fragmentation, and cell morphology were assessed to determine the toxicity of thiourea-reduced graphene oxide (T-rGO) on human lung cancer cells. The results revealed that GO and T-rGO were hazardous to cells in a dose-dependent trend. The viability of both <i>A. fumigatus</i> and <i>A. flavus</i> was affected by GO and T-rGO. The reactive oxygen species produced by T-rGO caused the death of <i>A. flavus</i> and <i>A. fumigatus</i> cells. This study highlighted the effectiveness of T-rGO as an antifungal agent. In addition, T-rGO was found to be more harmful to cancer cells than GO. Thus, T-rGO manifested great potential in biological and biomedical applications.