© 2021, Pleiades Publishing, Inc.Naphthoquinones harboring 1,4-naphthoquinone pharmacophore are considered as privileged structures in medicinal chemistry. In pharmaceutical industry and fundamental research, polyketide naphthoquinones were widely produced by heterologous expression of polyketide synthases in microbial chassis cells, such as Saccharomyces cerevisiae and Escherichia coli. Nevertheless, these cell factories still remain, to a great degree, black boxes that often exceed engineers’ expectations. In this work, the biotransformation of juglone or 1,4-naphthoquinone was conducted to generate novel derivatives and it was revealed that these two naphthoquinones can indeed be modified by the chassis cells. Seventeen derivatives, including 6 novel compounds, were isolated and their structural characterizations indicated the attachment of certain metabolites of chassis cells to naphthoquinones. Some of these biosynthesized derivatives were reported as potent antimicrobial agents with reduced cytotoxic activities. Additionally, molecular docking as simple and quick in silico approach was performed to screen the biosynthesized compounds for their potential antiviral activity. It was found that compound 11 and 17 showed the most promising binding affinities against Nsp9 of SARS-CoV-2, demonstrating their potential antiviral activities. Overall, this work provides a new approach to generate novel molecules in the commonly used chassis cells, which would expand the chemical diversity for the drug development pipeline. It also reveals a novel insight into the potential of the catalytic power of the most widely used chassis cells.