Eight styrylpyrylium tetrafluoroborate salts have been synthesized and fully optically characterized by UV-vis absorption and fluorescence steady-state/time-resolved spectroscopies. The new dyes exhibit strong emission bands with yellow-orange colours, depending on the substituents present in the structure. Notably, the Stokes shift recorded for some of them exceeds 100 nm, a very valuable feature for biological imaging. Four of them have been assayed as biological imaging agents by confocal laser scanning microscopy (CLSM) in the human hepatoma cell line Hep3B. It has been found that all the compounds efficiently stain intracellular structures which have been identified as mitochondria through colocalization assays with MitoView (a well-known mitochondrial marker) and using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a mitochondrial membrane potential uncoupler. Additionally, the potential ability of the studied dyes as cytotoxic drugs has been explored. The inhibitory concentration (IC50) against Hep3B was found to be in the range of 4.2 μM-11.5 μM, similar to other described anticancer drugs for the same hepatoma cell line. The combined features of a good imaging agent and potential anticancer drug make the family of the studied pyrylium salts good candidates for further theranostic studies. Remarkably, despite the extensive use of pyrylium dyes in several scientific areas (from photocatalysis to optics), there is no precedent description of a styrylpyrylium salt with potential theranostic applications.