Quantification of bacteria attached to sand grains is usually not possible without destroying biofilms or the sand structure. In situ quantification of autofluorescing or of fluorescence-carrying bacteria might be an alternative if fluorescence intensity (FI) was strong enough and could be related to cell numbers. For calibration, a Hele–Shaw cell was filled with sterile quartz sand and sectioned for inoculation with increasing cell densities of Escherichia coli strain HB101 K12 pGLO, overexpressing green fluorescent protein (GFP). Digital photographs of the single sections were taken under ultraviolet light at wavelength λ = 365 nm for fluorescence quantification by TotalLab Quant software. The FI was related to respective cell densities per quartz sand volume. Growth of GFP-labeled E. coli, suspended in Lysogeny broth (LB) medium (dissolved organic C = 6.8 g L−1), could thus be quantified noninvasively in capillary fringe (CF) laboratory systems simulating static or hydrodynamic conditions. Best growth conditions existed in the transition zone of the CF, where after 3 d the highest cell densities of 5.0 to 6.5 × 108 cells cm−3 were determined. The E. coli cells that grew attached as a biofilm in the different sections of the CF could also be quantified under flow-through conditions and after fluctuation of the water table. For reliable results, FI calibration must be performed in the same setup as used for experiments, and fluorescence characteristics of the bacterial strain should be well known. This noninvasive method using bright fluorescing bacteria can be advantageous for the determination of bacterial growth and biofilm formation in laboratory experiments with unsaturated soil under changing hydraulic conditions.