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Applications of plasma-induced biological effects via reactive oxygen and nitrogen species (RONS) make the non-thermal atmospheric-pressure plasma jets an appealing tool in biomedical fields. The presence of biological materials, especially as part of the electrode circuit, may change the plasma properties and impact on the production of RONS at the plasma–biomaterial interface. Effects of biomaterials on the production of hydroxyl radicals (OH) in a nanosecond pulsed, atmospheric-pressure plasma jet were investigated using a needle-to-plate electrode configuration with water, phosphate-buffered saline (PBS), or pigskin covering the ground plate. Driven by 200 ns, 7 kV pulses at 1 kHz, a helium plasma jet was generated between the hollow needle electrode and the biomaterial. Temporally resolved UV-visible imaging showed that the use of pigskin slowed down the streamer head propagation, whereas a more pronounced surface ionization wave was developed on the surface when water was used. The highest OH(A-X) emission above the biomaterial surface was observed using the PBS-covered electrode plate comparing to water or pigskin. Spatiotemporally resolved laser-induced fluorescence (LIF) showed that more OH was produced in the region near the needle electrode for both water and PBS, and the use of pigskin resulted in least OH production overall. In addition, measurements of H2O2 production in the liquid were used to determine the OH concentration in the vicinity of the biomaterial and agreed well with the relative OH-LIF measurements obtained at the gas–liquid interface for water and PBS.