Antagonism among bacteria is widespread and plays an important role in structuring communities. Inhibitory compounds can confer competitive advantage, but energetic trade-offs can result in non-transitive (i.e. ‘rock-paper-scissors’) interactions, ultimately allowing co-existence and community stability. Competition in sedimentary habitats is especially keen given high densities and attachment to inorganic particles. Because measuring trade-offs between bacterial species is challenging, much of our understanding of competitive interactions is based on theoretical modelling and simplified in vitro experiments. Our objectives were to determine (1) if interference competition occurs in microcosms mimicking in situ conditions; (2) whether the presence of sediment influences antagonistic interactions; and (3) if more complex assemblages alleviate or synergize interactions. Four sedimentary isolates, including antibiotic-producing, resistant and susceptible strains were incubated in porewater microcosms in 1-, 2- and 3-species combinations, both with and without natural sediments. Microcosms were sampled over 72 h to generate growth curves using quantitative PCR. Multiple growth attributes (growth rate, maximum density, lag time) were used to assess effects of treatment (species combinations) and environment (sediment vs porewater alone). Antimicrobial producers were more effective at inhibiting target species in microcosms that included sediment, in agreement with theory. We observed growth inhibition by antimicrobial-producing bacteria in both 2- and 3-species microcosms. However, the expected protection of sensitive bacterial strains by resistant strains was observed in only one (of four) 3-species combinations, thus the ‘rock-paper-scissors’ prediction was not fully supported. These results reinforce the notion that interspecies interactions are context-dependent, reliant on environmental conditions and the species involved.