<p>Species richness is often positively linked to ecosystem functioning. However, conventional approaches that manipulate richness frequently confound the richness effect with those of species composition. In this study, we quantitatively disentangled the independent effects of species richness and composition on biofilm productivity as a measure of ecosystem functioning. We constructed 300 independent richness gradients, each comprising 3–20 species, from a pool of 24 bacterial isolates spanning six taxonomic classes (Actinomycetia, Alphaproteobacteria, Bacilli, Betaproteobacteria, Gammaproteobacteria, and Sphingobacteriia). Our results revealed diverse richness–biofilm relationship forms, predominantly positive (68%), but also hump-shaped (16%), U-shaped (6%), null (6%), and negative (4%). When assessed individually, richness accounted for an average of 53.9% of the variation in biofilm growth, surpassing two measures of compositional variation—species-inherent ability (SIA) and species-dependent ability (SDA)—based on species’ biofilm-forming potential, which explained 9.6% and 14.4%, respectively. Collectively, richness, SIA, and SDA explained 73.4% of the variation. Notably, biofilm growth exceeded expectations in the mid-richness range (10–15 species). When richness and composition were assessed collectively across entire assemblages, richness explained 24.2% of the variation in biofilm growth, while SIA and SDA explained 12.7% and 28.7%, respectively. In contrast, species combination, assuming equal potential for all species, had only a marginal effect on biofilm growth. Our results demonstrate that species richness is a key, independent driver of biofilm growth, and that its effects are substantially underestimated when not properly separated from composition.</p>