The detection and inhibition of Clostridium botulinum by probiotics' cell-free supernatants, as a bio-preservation strategy, were investigated in some Egyptian fish-stuffs i.e., canned sardines, fesikh, moloha, renga, and anchovy. The conventional and multiplex polymerase chain reaction were used to detect C. botulinum, while the inhibitory and antioxidant capacity of four probiotics' cell-free supernatants were assessed using disc diffusion assay (DDA) and radical scavenging activity approaches, respectively. The results revealed that the C. botulinum was isolated from 41.6% of the examined products, where the type E. was the most predominant genotyping. The DDA showed that Lactobacillus bulgaricus EMCC 1102 and L. paracasei EMCC 1103 expressed the highest inhibitory action against C. botulinum type E. Meanwhile, L. bulgaricus EMCC 1102 has higher in vitro antioxidant activity (IC₅₀) than L. paracasei EMCC 1103, mostly due to its high bioactive constituent. Consequently, L. bulgaricus EMCC 1102 was used to control the growth of C. botulinum type E in stored anchovy fillets. L. bulgaricus EMCC 1102 supernatant showed a dose-dependent inhibitory effect on C. botulinum type E. These data provided an innovative perspective on the inhibitory potential of probiotics, as a bio-preservation strategy, to guarantee fish products safety for further applications in the food industry.