Due to their exceptional characteristics, nanomaterials are applied in multiple fields, including automotive, textiles or biomedical sectors, being Nanotechnology conceived as a Key Enabling Technology (KET). For this reason, the risks to health and environment posed by the special particularities of nanomaterial enabled products are critical to identify and control their associated hazards. Environmental research has focussed on elucidating and modelling of toxicokinetics and toxicodynamics of different types of nanomaterials in soil and aquatic organisms. In this regard, biofilms are considered as good bioindicators of contamination due to their ability to accumulate and react quickly to xenobiotics. Despite this, currently there is not any OECD validated protocol to study toxicokinetics and toxicodynamics of xenobiotics, and there is only one available study in the current literature where both parameters are analysed in these structures (Chaumet et al., 2019) The aim of this work was to set up the appropriate conditions to develop a protocol to study Multi-Component NanoMaterials (MCNMs) and High Aspect Ratio Nanoparticles (HARNs) tokicokinetics and toxicodynamics in biofilms. Pseudomonas putida, a Gram-negative bacterium that can be found in soils being involved in several relevant functions, was selected as model organism. Different conditions of temperature, surface material, incubation time etc. were tested in order to obtain significant biofilm biomass to perform toxicokinetic studies. Results showed that the incubation of bacterial cultures during 96 hours at 37°C, in polystyrene well plates coated with a polyelectrolyte lead to the highest amount of biofilm. For toxicodynamics studies, ROS production was selected as a parameter to be analysed using the CM-H2DCFDA dye. The appropriate experimental conditions were first set up with planktonic bacteria exposed to H2O2, and then applied in biofilms. This study has received funding from the European Union’s Horizon 2020 program (DIAGONAL, Grant Agreement no 953152). The DIAGONAL project aims to address existing gaps at risk assessment, risk management and risk governance levels providing new knowledge on multicomponent nanomaterials and high aspect ratio nanomaterials.