Degradation of residuals of chemical compounds after use has attracted the attention of environmental scientists. In this work, the degradation of such a toxic biocide compound of isothiazolin-3-one from an aqueous solution using a novel multi-pin thermally nonequilibrium atmospheric pressure plasma reactor is reported. This investigation examined the impacts of different operating conditions on the decomposition of isothiazolin-3-one. Subsequently, the reactive excited plasma species formed and participated during the degradation processes were examined by optical emission spectrometer (OES), chemical dosimetry using terephthalic acid (TA), and spectroscopic determination involving potassium titanium (IV) oxalate. The efficiency of degradation of the plasma-treated isothiazolin-3-one from an aqueous solution was examined by UV-Vis spectrometry. The mineralization of isothiazolin-3-one was further examined by high-performance liquid chromatography (HPLC), determining conductivity, total organic carbon (TOC), and pH of the isothiazolin-3-one aqueous solution. Finally, nontoxicity of the plasma-treated isothiazolin-3-one solution was investigated by seed sprouting and germination tests using Vigna radiata L. Wildzek (Mung bean). The experimental results exhibit that a higher degradation percentage of 96.01% was attained for the isothiazolin-3-one treated at a longer treatment time and higher potential of 25 min and 31 kV, respectively. This may be attributed to the formation of various reactive oxygen and nitrogen species (RONS) as confirmed by OES and other spectroscopic analyses. The value of pH and TOC is found to be decreased, whereas the value of electrical conductivity of the isothiazolin-3-one aqueous solution is increased linearly with an increase in treatment time and applied voltage. This indicates the degradation of isothiazolin-3-one molecules and the formation of various ionic species in the solution. The toxicity investigation reveals that the plasma-treated isothiazolin-3-one aqueous solution is nontoxic in nature and useful for sprouting and germination of the Mung bean seeds compared with the untreated one. Overall, it can be concluded that the plasma-assisted process is ideally needed in various food, pharmaceutical, and textile industries as a highly efficient treatment of wastewater.