The research was focused on in silico characterization and in vitro biological testing of the series of the compounds carrying a N-arylpiperazine scaffold. Their in silico investigation was based on the prediction of electronic, steric and lipohydrophilic features. The in vitro inhibitory impact of those molecules on a photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts was evaluated using the Hill reaction. Among the tested N-arylpiperazines, the most promising potential to inhibit the PET was found for 1-[3-(3-ethoxyphenyl- carbamoyl)oxy-2-hydroxypropyl]-4-(3-trifluoromethylphenyl)piperazin-1-ium chloride and 1-[3-(4-ethoxyphenylcarbamoyl)oxy-2-hydroxypropyl]-4-(3-trifluoromethylphenyl)piperazin-1-ium chloride. The current study discussed preliminary structure–photosynthesis-inhibiting activity relationships considering electronic, steric and lipophilic properties.