Microbial biofilms play an important role in the process of absorption and bioaccumulation of heavy metals in polluted aquatic environments, including hexavalent chromium [Cr(VI)] which is toxic and persistent. Although many studies have examined the potential of biofilms in bioremediation, information on the structural characteristics and bioaccumulation capacity of natural biofilms in industrial river ecosystems, particularly in Indonesia, is still limited. This study aims to examine the structural characteristics, composition of microbial communities, as well as the absorption and bioaccumulation capacity of biofilms against Cr(VI) in the Manyar River ecosystem, Gresik. The results of the analysis show that the formation of biofilms takes place through three main stages, namely early adhesion, growth, and cellular release. Treatment with Cr(VI) causes the appearance of new absorption bands at certain wavelengths indicating the presence of functional groups C–X, C–O, and C=C, as well as an increase in the intensity of the O–H group, indicating a modification of surface chemistry due to metal interactions. Based on community analysis, biofilms are dominated by Virgibacillus spp., Romboutsia spp., and Streptococcus spp., reflecting adaptation to polluted water conditions. The value of the distribution coefficient (Kd) for Cr(VI) ranges from 0.99–1.05, while the Bioaccumulation Factor (BAF) reaches 99.44–106.81 times, indicating the high ability of biofilms to accumulate Cr(VI). These findings fill the knowledge gap regarding the potential of natural biofilms from industrial river ecosystems in Indonesia as bioremediation agents of heavy metals, especially Cr(VI), and open up opportunities for sustainable application in the management of polluted water quality.