Photodynamic inactivation (PDI) is an attractive treatment modality for multidrug-resistant bacterial infections. The effectiveness of photosensitization by anionic photosensitizers such as erythrosine B can be further enhanced by the addition of biological or chemical molecules. This study aimed to investigate of the enhancement effect of acetic acid and chitosan on erythrosine-mediated PDI of Acinetobacter baumannii in planktonic and biofilm forms. The planktonic cell growth of three A. baumannii strains was subjected to PDI by using erythrosine B (50 µM) in 0.01% acetic acid and green laser light (530 nm) at fluence of 40 J/cm2. The phototoxic effect of erythrosine B (100 µM) in combination with chitosan (12.5 mg/ml) (in a solution of acetic acid) at fluence of 80 J/cm2 on biofilms was also evaluated. Finally, the cytotoxicity and phototoxicity of the mentioned mixture were assessed on human fibroblasts. Planktonic cells of all three studied A. baumannii strains were almost eradicated by erythrosine B-mediated PDI in the presence of acetic acid. Also, PDI combined with chitosan resulted in a marked decrease in the number of viable biofilm cells (> 3 log10 CFU). At the same experimental conditions, only 15% of the fibroblasts were photoinactivated. The results showed that PDI by using erythrosine B in acetic acid is very effective against A. baumannii planktonic cells and could eliminate them significantly. Also, chitosan enhanced the anti-biofilm efficacy of erythrosine B-mediated PDI against A. baumannii, suggesting that combination therapy may be useful in targeting biofilms.