The rise of drug-resistant pathogens necessitates innovative approaches like antimicrobial photodynamic inactivation (aPDI). This study examines the influence of surfactants and solvents on the efficacy of photosensitizers (PS) in aPDI. A key challenge in aPDI is the tendency of PS to aggregate in physiological environments, driven by intermolecular forces such as van der Waals interactions. Aggregation affects the photophysical and photochemical properties of PS, altering their light absorption and affecting the production of reactive oxygen species (ROS), specially singlet oxygen, essential for microbial inactivation. Phenothiazinium dyes, such as methylene blue (MB), exhibit significant self-aggregation, reducing their antimicrobial efficiency. The use of surfactants like sodium dodecyl sulfate (SDS) has proven effective in disrupting these aggregates, enhancing the availability of monomeric form and optimizing singlet oxygen production. Solvents such as ethanol and specialized mixtures like MIX (glycerol:ethanol:water) further reduce aggregation and extend singlet oxygen’s half-life, thus improving aPDI efficacy. Planar macrocycles such as porphyrins, chlorins, phthalocyanines exhibit similar behavior, and both surfactants and solvents can affect their aggregation behavior. It is important to note that while some PS exhibit reduced ROS production and aPDI action when aggregated, others show the opposite behavior, with enhanced ROS production and aPDI action in the aggregate form. This study underscores the importance of carefully selecting surfactants and solvents to maximize the therapeutic potential of PS for aPDI in combating various infections, specially drug-resistant ones.