The fluorescent dyes 9-aminoacridine (9-AA) and acridine orange (AO) are known mutagens that induce frameshift mutations in cells by intercalating between DNA bases. However, these chemicals can also affect other cellular components, such as proteins. In this study, we tested the ability of 9-AA and AO to induce heat shock in bacteria using the following methods: lux-biosensors based on Escherichia coli cells with the luxCDABE genes transcriptionally fused to heat shock-specific inducible promoters, RT-qPCR, and nanoDSF. We demonstrated that acridine dyes not only induce mutagenesis but also cause heat shock in bacterial cells. AO significantly reduced the melting temperature of proteins and strongly activated σ^E- and σ³²-dependent promoters, but not PluxC, which is activated by elevated temperatures via a different mechanism. In contrast, 9-AA weakly denatured the proteins and induced the σ^E-dependent promoter; however, it activated the σ³²-dependent promoters and PluxC, supporting the hypothesis that the σ³² heat shock response system is activated via hairpin RNA denaturation by 9-AA. The study on the application of lux-biosensors was hampered by the high general toxicity and luminescence shielding effect of AO, and RT-qPCR’s sensitivity was insufficient for detection of the response to 9-AA. Thus, methodologically, it is justified to conduct a comprehensive study of substances that cause heat shock or affect bioluminescence by both RT-qPCR and lux-biosensors.