The bioactivity of the most abundant sponges from three communities in a Mediterranean cave was assessed by Microtox® assay in two seasons, spring (June) and autumn (November). We quantified bioactivity as a proxy for the investment in production of biologically active substances, and we related sponge bioactivity to growth form, growth rates, and physical contacts of each species with other species. We established a threshold for classifying a species as bioactive based on a comparison between the results of the Microtox® and the sea urchin embryo toxicity bioassay. A total of 30 species were included in the study, of which 50% were bioactive in some community or season. Significant ecological (between communities) and seasonal variation in mean bioactivity was found. When sponge bioactivity was related to sponge growth shape, it was found that the encrusting species tended to be more toxic than the non-encrusting ones. There was a negative relationship between bioactivity and sponge growth, suggesting a trade-off in energy allocation to defence and to other biological functions. Furthermore, a negative correlation was found between bioactivity and positive associations with other species. These results highlight the important role of chemically-mediated interactions in cave communities.