A novel series of benzimidazole type of Fluconazole analogues were synthesized and characterized by (1)H NMR, (13)C NMR, IR, MS and HRMS spectra. All the new compounds were screened for their antimicrobial activities in vitro by two-fold serial dilution technique. The bioactive evaluation showed that 3,5-bis(trifluoromethyl)phenyl benzimidazoles gave comparable or even stronger antibacterial and antifungal efficiency in comparison with reference drugs Chloromycin, Norfloxacin and Fluconazole. The combination of 2,4-difluorobenzyl benzimidazole derivative 5m and its hydrochloride 7 respectively with antibacterial Chloromycin, Norfloxacin or antifungal Fluconazole showed better antimicrobial efficiency with less dosage and broader antimicrobial spectrum than the separated use of them alone. Notably, these combined systems were more sensitive to Fluconazole-insensitive Aspergillus flavus and methicillin-resistant MRSA.