Three-dimensional (3D)-printing technology is instrumental in creating devices for biological applications, including the exploitation of cold atmospheric plasma (CAP). CAP, a partially ionized gas that functions at ambient temperatures, serves as a safe, inexpensive, and effective tool for the inactivation of various pathogens on different surfaces. In this study, we compared three different 3D-printed devices with respect to their ability to provide optimized CAP compositions effective against select respiratory viruses (SARS-CoV-2, influenza virus, adenovirus, and rhinovirus) and the bacterium Pseudomonas aeruginosa, which is associated with serious lung diseases. The transmission of respiratory pathogens via surface contamination may pose a serious health threat, thus highlighting the biological importance of the current study. The properties of a prototype 3D-printed CAP-generating device and two optimized versions were characterized by detecting reactive oxygen and nitrogen species (RONS) in a gaseous environment via infrared spectroscopy and analyzing the composition of the reactive compounds. The virucidal effects of CAP were examined by determining virus infectivity and particle integrity. The bactericidal effect was documented by viability testing and visualization via transmission electron microscopy. The findings indicate that optimization of the 3D-printed devices for CAP production yielded an environment with relatively high amounts of RONS (O3, N2O, NO2, and H2O2), reducing the exposure time required for inactivation of respiratory pathogens by approximately 50%. In addition to reducing infectivity and viability, CAP treatment led to the destruction of viral nucleic acids and physical damage to bacterial cells. Owing to its flexibility and easy implementation, optimized CAP generated by 3D-printed devices provides an attractive inactivation method adaptable for different biological applications, including surface decontamination from viral and bacterial pathogens.