The increase in pathogen levels in seawater threatens the safety of entire aquatic ecosystems. Foodborne pathogens can potentially accumulate in shellfish, especially in filter feeders such as bivalves, requiring an efficient depuration process before consumption. Alternative approaches to promote a cost-efficient purge at depuration plants are urgently needed. A small prototype pulsed ultraviolet (PUV) light recirculation system was designed, and its depuration potential was tested in a seawater matrix artificially contaminated with high levels of microbial pathogensEscherichia coli,Staphylococcus aureus,Salmonella typhimurium,Bacillus cereusandCandida albicans. The analysis of treatment parameters including voltage, number of pulses and duration of treatment was performed to ensure the highest reduction in contaminant levels. Optimal PUV disinfection was attained at 60 pulses/min at 1 kV for 10 min (a UV output of 12.9 J/cm2). All reductions were statistically significant, and the greatest was observed forS. aureus(5.63 log10), followed byC. albicans(5.15 log10),S. typhimurium(5 log10),B. cereus(4.59 log10) andE. coli(4.55 log10). PUV treatment disrupted the pathogen DNA with the result thatS. aureus,C. albicansandS. typhimuriumwere not detectable by PCR. Regulations were reviewed to address the applicability of PUV treatment as a promising alternative to assist in the reduction of microbial pathogens at depuration plants due to its high efficiency, short treatment period, high UV dose and recirculation system as currently employed in shellfish depuration plants.