Understanding the influence of attached microbial biomass on water flow in variably saturated soils is crucial for many engineered flow systems. So far, the investigation of the effects of microbial biomass has been mainly limited to water‐saturated systems. We have assessed the influence of biofilms on the soil hydraulic properties under variably saturated conditions. A sandy soil was incubated with Pseudomonas Putida and the hydraulic properties of the incubated soil were determined by a combination of methods. Our results show a stronger soil water retention in the inoculated soil as compared to the control. The increase in volumetric water content reaches approximately 0.015 cm³ cm⁻³ but is only moderately correlated with the carbon deficit, a proxy for biofilm quantity, and less with the cell viable counts. The presence of biofilm reduced the saturated hydraulic conductivity of the soil by up to one order of magnitude. Under unsaturated conditions, the hydraulic conductivity was only reduced by a factor of four. This means that relative water conductance in biofilm‐affected soils is higher compared to the clean soil at low water contents, and that the unsaturated hydraulic conductivity curve of biofilm‐affected soil cannot be predicted by simply scaling the saturated hydraulic conductivity. A flexible parameterization of the soil hydraulic functions accounting for capillary and noncapillary flow was needed to adequately describe the observed properties over the entire wetness range. More research is needed to address the exact flow mechanisms in biofilm‐affected, unsaturated soil and how they are related to effective system properties.