Starting from sissotrin (1), a natural isoflavonoid isolated from Trifolium baccarinii (Fabaceae), one new semisynthetic derivative, 6-nitrobiochanin A (1b) and two known derivatives, 8-nitrobiochanin A (1a) and 2",3",4",6"-tetraacetylsissotrin (1c) have been obtained after performing nitration and acetylation reactions. Their structures were assigned after interpretation of their spectrometric (HR-ESI-MS) and spectroscopic (NMR 1D and 2D) data and by comparison with those reported in the literature. The substrate as well as the semisynthetic derivatives were evaluated for their antibacterial activities against six strains. The results reveal that they are inactive or weakly active on the strains tested with the exception of 8-nitrobiochanin A (1a) which showed moderate activity (MIC = 62.5 μg∙mL−1) on Staphylococcus aureus ATCC 43300.