Ballast water is one of the main vectors for the introduction of exotic species to coastal ecosystems. The D-2 standard of the International Maritime Organization (IMO) sets limits to the permitted maximum concentration of living organisms in ballast water to prevent marine invasions. This study evaluated the compliance with the D-2 standard of the ballast water of ships arriving to Chilean ports in 2017. Ballast water samples were collected from twelve ships at the ports of Valparaíso, San Vicente, and Calbuco. The results revealed high non-compliance with the D-2 standard due to high concentrations of plankton in ballast water. A significant diversity of phytoplankton, comprising diatoms and dinoflagellates, was identified and among the zooplanktonic organisms, predominantly marine invertebrates. Physicochemical parameters such as oxygen, temperature, and pH were variable, but within ranges that sustain aerobic life. No pathogens were detected in the microbiological samples, although enterococci and Escherichia coli were found within permissible limits in some vessels. These findings highlight the inefficacy of current ballast water treatments and the need to improve policies and regulations to prevent biological invasions and protect Chile’s marine biodiversity. It is recommended to implement more rigorous and periodical monitoring to strengthen international regulations in ballast water management.