Bacterial interference studied by means of agar methods has shown a decreased number of inhibitory alpha-haemolytic Streptococci among otitis-prone children. Additional information was gained regarding the interplay between alpha-haemolytic Streptococci (AHS) and otitis media (OM) pathogens by comparing the bacterial interference in broth with the interference activity studied using agar overlay methods. We found, that non-typeable Haemophilus influenzae (NTHI) and Moraxella catarrhalis are readily inhibited by AHS in broth. Streptococcus pneumoniae was more bacteriostatically inhibited. If two OM pathogens were inoculated simultaneously, an isolate of AHS with poor inhibitory activity was not able to inhibit the growth, in contrast to an isolate of AHS with good inhibitory activity. The initial amount of AHS inoculated with M. catarrhalis seemed to play a decisive role with respect to the inhibitory activity. M. catarrhalis developed reduced susceptibility against AHS both in vivo and in vitro. In vivo studies showed that children with secretory otitis media had fewer isolates of AHS in their nasopharynx with the ability to inhibit all the test pathogens than healthy children (p < 0.001). Although the factor(s) responsible for the inhibitory activity have thus far not been defined, we could exclude low pH and nutrition depletion as the inhibitory mechanism of AHS with good inhibitory activity.