Three series of genistein derivatives with heterocycles were prepared, in which genistein and heterocyclic moieties were separated by 2-carbon, 3-carbon and 4-carbon spacers. Among the 33 compounds we prepared 11 of them (2c and 5a-j) are reported for the first time, while the preparation of 2a,b, 3a-j and 4a-j was reported in our recent paper. All the derivatives were screened for antibacterial (Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas fluorescence) and antifungal (Aspergillus niger, Candida albicans and Trichophyton rubrum) activities by MTT method. Among the compounds tested, 4a, 4e, 4f, 4h, 5e and 5f exhibited good antibacterial activities while 4a also showed notable antifungal activity. Especially, 5f exhibited stronger antibacterial activity against B. subtilis and S. aureus comparable to positive controls.