Background/aim: This study was designed to evaluate the effect of antimicrobial photodynamic treatment (APDT) in a biofilm model using combinations of various dyes (rose bengal, riboflavin, and methylene blue) as photosensitizers and light sources (LED and UVA) against staphylococcal and candidal biofilms. Materials and methods: Sterile microtiter plates were used for the development and quantification of the biofilms. APDT was carried out using combinations of the light sources and dyes. The percentage of the growth inhibition was then calculated using a spectrophotometer. The broth media in the wells were aspirated, wells were stained with crystal violet, and optical density values were measured spectrophotometrically. SEM analysis of the impact of APDT on bacterial and fungal biofilms was also performed. Results: The experiments showed that the most efficacious combination was red LED + methylene blue against both staphylococcal and candidal biofilms. A marked inhibition (45.4%) was detected on both C. albicans and C. parapsilosis biofilms. Red LED + methylene blue was also effective on S. aureus and S. epidermidis biofilms. SEM images suggested that the number of adherent cells and biofilm mass were markedly reduced after APDT treatment. Conclusion: Although the results of this study indicated the in vitro efficacy of APDT, it might also be a promising technique for the control of biofilm growth within intravenous catheters.