Background The potential synergism of spiramycin-metronidazole in combination against Aggregatibacter actinomycetemcomitans, a major pathogen in human periodontitis, peri-implantitis, and some non-oral infections, was studied in vitro in comparison to combinations of amoxicillin-metronidazole and ciprofloxacin-metronidazole. Methods Minimal inhibitory concentrations (MICs) for spiramycin, amoxicillin, ciprofloxacin, and metronidazole were determined individually against four periodontal A. actinomycetemcomitans clinical isolates. Synergism testing for the combinations of spiramycin-metronidazole, amoxicillin-metronidazole, and ciprofloxacin-metronidazole was performed with gradient diffusion strips for each antibiotic pair placed onto A. actinomycetemcomitans-inoculated Haemophilus test medium in a cross formation at the intersection of each of their individual MICs against A. actinomycetemcomitans .Fractional inhibitory concentration index values assessed the antibiotic interactions. Results Spiramycin and metronidazole individually had poor antimicrobial activity against A. actinomycetemcomitans. However, lower MIC values and less A. actinomycetemcomitans resistance were found for both antibiotics when tested in combination. Spiramycin-metronidazole synergism was additionally detected against one A. actinomycetemcomitans clinical isolate. In comparison, combinations of amoxicillin-metronidazole and ciprofloxacin-metronidazole exhibited antimicrobial synergism against all four A. actinomycetemcomitans strains. Conclusions Spiramycin and metronidazole exerted greater in vitro antimicrobial activity in combination than individually against A. actinomycetemcomitans. Antimicrobial synergism between spiramycin and metronidazole was also found with one A. actinomycetemcomitans clinical isolate, representing the first detection of spiramycin-metronidazole synergism against A. actinomycetemcomitans. However, antimicrobial synergism against A. actinomycetemcomitans was less frequently detected with spiramycin-metronidazole as compared to combinations of amoxicillin-metronidazole or ciprofloxacin-metronidazole. These findings may help clinicians in the selection of effective antimicrobial therapies against oral and non-oral infections involving A. actinomycetemcomitans.