This work evaluated the genotoxic potential of the soluble organic material (SOM) extracted from the particulate matter (PM) emitted by an automotive diesel engine. The engine was modified to operate with a home-made multipoint-port injection system to substitute 10% of ultralow-sulfur diesel (ULSD) fuel in energy basis by hydrous ethanol (h-Et) or n-butanol (n-Bu) injected into the manifold during the intake stroke. A low engine load mode named M4 (43 N·m at 2410 min−1) and a medium-load mode M2 (95 N·m at 2410 min−1) were selected from the vehicle homologation cycle. PM was collected with a stainless steel filter located 1.5 m downstream the exhaust manifold. The SOM of the PM was extracted to evaluate the genotoxic activity on human lymphocytes using the comet assay. Results indicated that independently of the mode, the SOM coming from alcohols led more genotoxicity than ULSD, following the order h-Et > n-Bu > ULSD. The low engine load operation exhibited much more deoxyribonucleic acid (DNA) damage than mode M2, especially the PM produced by hydrous ethanol port-injection. Although further research is still necessary, these findings suggest that the biology activity of the SOM coming from alcohols PM could be a barrier for the implementation of alcohol port-injection technology.