Bioaccumulation of perylene in Escherichia coli and Staphylococcus aureus was visualized and quantified in real time with high sensitivity at high temporal resolution. For the first time, single-molecule fluorescence microscopy (SMFM) with a microfluidic flow chamber and temperature control has enabled us to record the dynamic process of perylene bioaccumulation in single bacterial cells and examine the cell-to-cell heterogeneity. Although with identical genomes, individual E. coli cells exhibited a high degree of heterogeneity in perylene accumulation dynamics, as shown by the high coefficient of variation (C.V = 1.40). This remarkable heterogeneity was exhibited only in live E. coli cells. However, the bioaccumulation of perylene in live and dead S. aureus cells showed similar patterns with a low degree of heterogeneity (C.V = 0.36). We found that the efflux systems associated with Tol C played an essential role in perylene bioaccumulation in E. coli, which caused a significantly lower accumulation and a high cell-to-cell heterogeneity. In comparison with E. coli, the Gram-positive bacteria S. aureus lacked an efficient efflux system against perylene. Therefore, perylene bioaccumulation in S. aureus was simply a passive diffusion process across the cell membrane.