Four series of novel pyridine derivatives (17a–i, 18a–i, 19a–e, and 20a–e) were synthesized and their antimicrobial activities were evaluated. Of all the target compounds, almost half target compounds showed moderate or high antibacterial activity. The 4‐F substituted compound 17d (MIC = 0.5 µg/mL) showed the highest antibacterial activity, its activity was twice the positive control compound gatifloxacin (MIC = 1.0 µg/mL). For fungus ATCC 9763, the activities of compounds 17a and 17d are equivalent to the positive control compound fluconazole (MIC = 8 µg/mL). Furthermore, compounds 17a and 17d showed little cytotoxicity to human LO2 cells, and did not show hemolysis even at ultra‐high concentration (200 μM). The results indicate that these compounds are valuable for further development as antibacterial and antifungal agents.