Kefiran is a functional fermented milk product traditionally used for its beneficial probiotic properties. It exhibits antimicrobial, antioxidant, anti-inflammatory anticancer and different health promoting characteristics. Although kefiran showed potential effects against many cancer cell lines, little information is present in the literature on its effect against cervical and hepatocellular carcinoma as well as on zebrafish embryos. The study aimed at investigating the cytotoxicity (in human cervical and hepatocellular carcinoma cell lines) and developmental toxicity (in zebrafish embryos) of kefiran produced by the fermentation of Lactobacillus kefiranofaciens. Cervical and hepatocellular cancer cells were exposed to serial concentrations of kefiran to evaluate its cytotoxic activities. Further biological effects of kefiran on the mortality and developmental abnormalities of zebrafish embryos were investigated. Results showed that kefiran significantly affected the viability of both tested cancer cell lines in a dose-dependent manner with IC50 values of 358.8 ± 1.65 and 413.5 ± 1.05 μg/ml for HeLa and HepG2 cells, respectively. Furthermore, kefiran adversely affected the morphological characteristics of the cells. Kefiran extract was much safer for zebrafish embryos and no mortality was observed up to 100 μg/ml, whereas the LC50 value (≥ 279.76 μg/ml) was also very high. Moreover, no developmental toxicity was observed up to 100 μg/ml concentration. Conclusively, microbially-produced kefiran showed anticancer properties in two tested human cancer cells, while its safer profiles in animals (zebrafish embryos) poses it as potential anticancer agent which does not affect normal tissue growth.