The dripper clogging due to the development of biofilm can reduce the benefits of micro-irrigation technology implementation using reclaimed wastewater. The narrow cross-section and labyrinth geometry of the dripper channel enhance the fouling mechanisms. The aim of this study was to evaluate the water distribution and biofouling of drip irrigation systems at the field scale during irrigation with treated wastewater. Six 100 m lines of commercial pipes with two pressure-compensating dripper types (flow rate, Q, of 0.65 L h−1 and 1.5 L h−1, respectively) were monitored for four months. Different zones along the pipes were selected to evaluate the influence of hydrodynamical conditions (Reynolds number = 5400 to 0) on biofouling. Destructive methods involving the biofilm extraction by mechanical means, showed little biofilm development without significant differences in dry and organic matter content in function of the sampling location along the pipe or dripper flow rate (Q0.65 and Q1.5). These results were confirmed by non-destructive methods, such as optical coherence tomography, that nevertheless showed that biofouling concerned 15–20% of the total dripper labyrinth volume. Total organic carbon monitoring and its composition (by three-dimensional excitation and emission matrix fluorescence microscopy) showed that the biofilm did not significantly influence the organic matter nature. Our results indicated that the biological activity and biofilm development in irrigation systems were more affected by the environmental conditions, particularly water temperature, rather than flow conditions. This confirmed that treated wastewater with low organic content can be used in micro-irrigation systems without significant loss of efficiency, even in conditions requiring intensive irrigation, such as the Mediterranean climate.