Aims: The current work determined the cytotoxicity of the symbiont bioluminescent bacteria isolated from fresh ink of the squid, Photololigo duvaucelii for human colon cancer cell lines HT-29 and HCT116 and normal human dermal fibroblasts. Methodology and results: The crude sterile culture supernatants of the bacterial isolates grown in marine broth containing 2.8% of NaCl were tested for their cytotoxic activities for HT-29 and HCT-116 human colon cancer cells and the normal human dermal fibroblasts HDFn using Presto Blue™ Resazurin Assay. Zeocin served as the positive control. The cytotoxicity index profiles of all culture supernatants and negative control (marine broth with 2.8% NaCl) for HDFn suggest non-toxicity to the cells, whereas most culture supernatants were observed to be cytotoxic to the two colon cancer cell lines. The culture supernatants of the isolates were found to be more cytotoxic for the HT-29 colon cancer cells than to the HCT-116 colon cancer cells. At the same time, the IC50 values showed that 85% (17/20) and 40% (8/20) of the culture supernatants tested were significantly lower, hence more potent than zeocin for HT-29 and HCT-116, respectively (p < 0.05). The rest were equally potent (p > 0.05). The 16S rRNA gene sequence analysis of the bioluminescent isolates studied revealed that they have a 97-99% similarity identity with Photobacterium leiognathi. Conclusion, significance and impact of study: This may be the first report on the cytotoxic activities on cancer cells of P. leiognathi from the Philippine squid and suggests the potential use of the secondary metabolites of these bioluminescent bacteria as anti-cancer agents.