Bacteria mostly live in collectives, where social interactions are prevalent. In biofilms, groups of surface-attached matrix-bound microbial cells exist and interact. Recent studies demonstrate that experimental evolution can be applied to biofilms to investigate and monitor the alterations in social interactions within the spatial structures. Here, experimental evolution approaches were employed to examine the evolutionary interplay of social activities in relation to the production of the biofilm matrix in pellicle biofilms of Bacillus subtilis, a plant beneficial soil-dwelling bacterium. The significant discoveries in this work are: 1)Biofilms are adequate models to study and monitor the origin and development of social interactions as these provide a tractable system to understand the impact of spatial distribution on microbial evolution. 2)Experimental evolution of biofilms facilitates diversification in clonal populations and results in complex interactions involving cooperative and exploitative behaviours. 3)Evolution of competitive interactions between matrix producers and non-producers results in an enhanced competitive advantage of the non-producers in the biofilm via the release of bacteriophages. 4)The exopolysaccharide (EPS) and protein components (TasA) of B. subtilis biofilms are costly public goods that facilitate the division of labour that is optimal at the genotypic level. 5)Genetic division of labour of costly public goods, EPS and TasA, is diminished by the arose of individuality during experimental evolution that selects for novel biofilm traits. 6)Experimental evolution of matrix producers in the presence of cheaters leads to an alteration in phenotypic heterogeneity that creates an anti-cheating mechanism within the biofilm. The findings presented here demonstrate how interactions in spatially structured environments become diverse, adaptive, maintained, and/or developed into unique biofilm traits, thus, contributing to Sociomicrobiology.