Conjugated polymers are versatile materials with promising applications as light-activated antibacterial agents. This work investigated the photoantimicrobial effect of conjugated polymer nanoparticles (CPNs) composed of poly(2.5-di(hexyloxy)cyanoterephthalylidene) (CN-PPV) prepared under different conditions via a nanoprecipitation method. The stabilization of CN-PPV CPNs with the surfactant polysorbate 20 (tween® 20) increased the CN-PPV product yields and resulted in the formation of nanoparticles of diminished size with slightly negative charge; which did not affect the cell viability of S. aureus and E. coli in the absence of light. Altering the ratio of the organic to aqueous phase from 1 : 10 to 2 : 10 (tetrahydrofuran – THF:water) led to the formation of CPNs of smaller size that presented increased photostability compared to that of 1 : 10 counterparts. All CPNs were capable of generating reactive oxygen species (ROS) under illumination (450 nm). In addition, CPNs produced with a 2 : 10 THF : H₂O ratio presented the highest photoantimicrobial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. Overall, by changing the CPN preparation conditions, CPNs of the same conjugated polymer having remarkably different properties, including greater photostability and more effective microorganism inactivation following photoexcitation, can be generated.