Background: Antimicrobial photodynamic inactivation (APDI) is a new therapeutic modality which needs more precision during application due to the possibility of exposure of bacteria to sub-lethal doses (sAPDI). In this study, we aimed to evaluate the effect of sAPDI on Pseudomonas aeruginosa quorum sensing (QS) and c-di-GMP signaling which are important virulence factor regulatory systems. Methods: Biofilm formation, pyoverdine, pyocyanin and protease production of P. aeruginosa was evaluated before and after a single sAPDI treatment with 0.8 mM methylene blue (MB) plus 1, 2, and 5-min irradiation with red laser light. Fluorescent lasB, rhlA, pqsA, and cdrA reporters of P. aeruginosa PAO1 and P. aeruginosa ΔmexAB-oprM were treated individually with sAPDI and the regulatory signals were detected. The gene expressions were also assessed after sAPDI using quantitative real-time PCR analysis. Results: Morphological observations and molecular assessments indicated that sAPDI with 0.8 mM MB along with 2- and 5-min irradiation led to an increase in the expression of the Las QS system and c-di-GMP signaling, while 1 min irradiation revealed dissimilar results (increase in lasB expression and decrease in c-di-GMP levels). Expression of rhlA and pqsA did not change in response to sAPDI. Further, a severe lethal effect of sAPDI was observed in P. aeruginosa ΔmexAB-oprM as compared with the wild type strain, whilst there was no difference in QS and c-di-GMP levels as detected by reporters between treated and untreated samples. Conclusion: The results suggest that sAPDI affects QS and c-di-GMP signaling inP. aeruginosa in a time-dependent manner.