Microscale cultivation systems are important tools to elucidate cellular dynamics beyond the population average and understand the functional architecture of single cells. However, there is scant knowledge about the bias of different microcultivation technologies on cellular functions. We therefore performed a systematic cross-platform comparison of three different microscale cultivation systems commonly harnessed in single-cell analysis: microfluidic non-contact cell traps driven by negative dielectrophoresis, microfluidic monolayer growth chambers, and semi-solid agarose pads. We assessed the specific single-cell growth rates, division rates and morphological characteristics of single Corynebacterium glutamicum cells and microcolonies as a bacterial model organism with medical and biotechnological relevance under standardized growth conditions. Strikingly, the specific single-cell and microcolony growth rates, μmax, were robust and conserved for several cell generations with all three microcultivation technologies, whereas the division rates of cells grown on agarose pads deviated by up to 50% from those of cells cultivated in negative dielectrophoresis traps and monolayer growth chambers. Furthermore, morphological characteristics like cell lengths and division symmetries of individual cells were affected when the cells were grown on agarose pads. This indicated a significant impact of solid cultivation supports on cellular traits. The results demonstrate the impact of microcultivation technology on microbial physiology for the first time and show the need for a careful selection and design of the microcultivation technology in order to allow unbiased analysis of cellular behavior.