Antibiotic-resistant microorganisms have become a global concern, and the search for alternative therapies is very important. Photodynamic therapy (PDT) consists of the use of a nontoxic photosensitizer (PS), light, and oxygen. This combination produces reactive oxygen species and singlet oxygen, which can alter cellular structures. Methylene blue (MB) is a substance from the phenothiazine class often used as a PS. In this work, to facilitate the PS contact within the wounds, we have used Design of Experiments 2(3) plus central point to develop functional polymeric systems. The formulations were composed by poloxamer 407 [15.0, 17.5, or 20.0% (w/w)], Carbopol 934P [0.15, 0.20, or 0.25% (w/w)], and MB [0.25, 0.50, or 0.75% (w/w)]. The sol gel transition temperature, flow rheometry, in vitro MB release, and ex vivo study of MB cutaneous permeation and retention were investigated. Moreover, the evaluation of photodynamic activity was also analyzed by in vitro degradation of tryptophan by singlet oxygen and using Artemia sauna. The determination of the gelation temperature displayed values within the range of 25-37 degrees C, and the systems with better characteristics were subjected to rheological analysis and in vitro release profiling. The 20/0.15/0.25 formulation showed the best release profile (42.57% at 24 h). This system displayed no significant skin permeation (0.38% at 24 h), and the photooxidation of tryptophan test showed the production of reactive species of oxygen. The toxicity test using A. salina revealed that the MB associated with the light increased the mortality rate by 61.29%. Therefore, investigating the PDT efficacy of the functional polymeric system containing MB will be necessary in the future.