This study investigated the occurrence and transport of four isothiazolinone-type biocides from commercial products to wastewater treatment plants (influents, sludges, and effluents) and to natural emissaries (upstream and downstream the wastewater treatment plants) in Romania. All four biocides were determined in personal care and household products, with the highest concentration of 76.4 µg/L OIT (2-octyl-4-isothiazolin-3-one). For environmental samples, three of the four isothiazolinones were determined, CMI (5-chloro-2-methyl-4-isothiazolin-3-one) being the prominent compound for water samples. The maximum concentration of 84.0 µg/L in influent, 122 µg/L upstream, and 144 µg/L downstream the wastewater treatment plants were obtained for CMI. Unlike water samples, in the sewage sludge samples, OIT proved to be the dominant compound, with concentration up to 5.80 µg/g d.w. The extremely high levels of isothiazolinone determined in different WWTPs from Romania may be due to the COVID-19 pandemic situation, during which a much larger amount of cleaning, hygiene, and personal care products was used. The isothiazolinone-type biocides were readily removed from the influents of the five WWTPs, with the mean removal rate up to 67.5%. The mean mass loading value for the targeted biocides based on influent was 20.4 μg/day/1000 people, while the average environmental emissions were 6.93 μg/day/1000 people for effluents. The results obtained for riverine water combine with statistical analysis showed that the anthropogenic activities are the major contamination sources of the surface waters. Preliminary ecological risk evaluation showed that BIT (1,2-benzisothiazol-3(2H)-one), OIT, and CMI could pose a very high risks to different aquatic species living in the receiving aquatic environments.