In the context of biofilm development, detachment is of practical importance when placed in a biofilm management perspective. The objective of the present study was to examine biofilm structure and biofilm detachment under controlled conditions for two distinct microorganisms grown under constant shear conditions. Detached biofilm biomass was regularly collected and analysed over the course of 72 h biofilm growth by Pseudomonas putida and Pseudomonas fluorescens cells, and biofilm structural development assessed using confocal microscopy. The two Pseudomonas spp., which had very similar specific growth rates in planktonic culture, presented notably different characteristics in terms of biofilm morphology but their detachment behaviours over time were very similar. These findings underline the intrinsic complexity of the detachment phenomenon.