Spatial connectivity and dispersal are key drivers of metacommunity assembly, shaping biodiversity patterns across landscapes. However, changes in connectivity, such as habitat fragmentation, pose a significant threat to these patterns and the maintenance of ecosystem biodiversity and functionality. In this study, we evaluated the effects of habitat fragmentation on planktonic microbial communities by analyzing taxonomic and functional diversity responses. We conducted a mesocosm experiment consisting of two phases: assembly phase and fragmentation phase. During the assembly phase, we established three metacommunity networks, each following the same centrality-isolation gradient. In the fragmentation phase, we imposed three fragmentation treatments: control (no fragmentation), modular, and strong fragmentation. Water samples were collected from all mesocosms initially, after the assembly phase and after the fragmentation phase. Prokaryotic biodiversity was assessed using DNA metabarcoding, while microbial functional diversity was evaluated using carbon substrate utilization profiles. We hypothesize a positive relationship between taxonomic alpha diversity and functional diversity, with both decreasing in the most isolated mesocosms. However, fragmentation is expected to modify taxonomic and functional diversity patterns also mismatching their relationships. This study highlights the pivotal roles of spatial connectivity and habitat fragmentation in shaping community assembly at both taxonomic and functional levels. By integrating multiple approaches, it aims to provide a deeper understanding of biodiversity patterns under varying landscape connectivity scenarios.