The microbial world represents a phenomenal diversity of microorganisms from different kingdoms of life, which occupy an impressive set of ecological niches. Most, if not all, microorganisms once colonize a surface develop architecturally complex surface-adhered communities, which we refer to as biofilms. They are embedded in polymeric structural scaffolds and serve as a dynamic milieu for intercellular communication through physical and chemical signalling. Deciphering microbial ecology of biofilms in various natural or engineered settings has revealed coexistence of microorganisms from all domains of life, including Bacteria, Archaea, and Eukarya. The coexistence of these dynamic microbes is not arbitrary, as a highly coordinated architectural setup and physiological complexity show ecological interdependence and myriads of underlying interactions. In this review, we describe how species from different kingdoms interact in biofilms and discuss the functional consequences of such interactions. We highlight metabolic advances of collaboration among species from different kingdoms, and advocate that these interactions are of great importance and need to be addressed in future research. Since trans-kingdom biofilms impact diverse contexts, ranging from complicated infections to efficient growth of plants, future knowledge within this field will be beneficial for medical microbiology, biotechnology, and our general understanding of microbial life in nature.