In Japan, Neisseria gonorrhoeae, a sexually transmitted pathogen, has recently shown significant resistance to various antimicrobial agents. In this study, a checkerboard method was utilized to investigate the in vitro activities of cefixime (CFIX), cefteram (CFTM), or amoxicillin (AMPC) in combination with azithromycin (AZM) against 25 clinical isolates of N. gonorrhoeae. Synergy, defined as a fractional inhibitory concentration (FIC) index of less than or equal to 0.50, was observed in 32% of isolates with CFIX+AZM, 12% of isolates with CFTM+AZM, and 4% of isolates with AMPC+AZM. Moreover, partial synergy, defined as an FIC index of greater than 0.50 and less than 1, was observed in 44% of isolates with CFIX+AZM, 68% of isolates with CFTM+AZM, and 52% of isolates with AMPC+AZM. In particular, as a result of the combination of CFIX and AZM, for all isolates, significant reductions were observed in the median CFIX minimum inhibitory concentration (MIC; from 0.25 to 0.008 microg/ml; P < 0.0001) and the median AZM MIC (from 0.12 to 0.03 microg/ml; P < 0.0001). However, antagonism, defined as an FIC index of greater than 1, was observed in only 4% of the isolates with both CFIX+AZM and CFTM+AZM, while it was seen in 12% of the isolates with AMPC+AZM. To our knowledge, this is the first study to demonstrate that the in vitro activity of CFIX against N. gonorrhoeae can be significantly enhanced in combination with AZM.